Identification and Functional Characterization of a Novel Activation Cascade of the KLK Family in Seminal Plasma
by
Nashmil Emami
Submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto
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Identification and Functional Characterization of a Novel Activation Cascade of the KLK Family in Seminal Plasma
Nashmil Emami Doctor of Philosophy 2009 Department of Laboratory Medicine and Pathobiology University of Toronto
ABSTRACT
Proteolytic processes are often mediated by highly orchestrated cascades, through
which protease enzymes function coordinately to ensure a stepwise activation. This thesis
presents experimental data which supports and complements the previously postulated
mechanism of KLK (kallikrein-related protease) activation through proteolytic cascades.
Further examination of the seminal KLK cascade has revealed several of its key (patho)
physiological roles in human reproductive system.
Multiple members of the seminal KLK cascade, in particular KLK14, were shown
to play a pivotal role in regulating semen liquefaction. The cascade was further shown to
be tightly regulated through a series of highly orchestrated feedback loops, to prevent
deleterious effects due to aberrant protease activation. Accordingly, a strong association
was observed between the expression level of several seminal KLKs, delayed liquefaction, and other markers of semen quality, including semen hyperviscosity.
Furthermore, a strong association was found between delayed liquefaction and abnormal
sperm motility. Therefore, dysregulated KLK expressions and/or activities were proposed
as an underlying cause of male subfertility.
Finally, this thesis has provided initial insights into a novel potential function of
multiple members of the seminal KLK cascade in activation of the key immune-deviating
ii
agent, TGFβ1, in seminal plasma. TGFβ1 activation is postulated to be mediated directly through complete fragmentation or indirectly through partial cleavage and conformational changes of the LAP propeptide motif of the latent TGFβ1. KLK- mediate proteolytic cleavage of the TGFβ1 binding protein, LTBP1, is also suggested as a potential physiological mechanism for release of the membrane-bound latent TGFβ1.
Overall, the data provided here may suggest a common regulatory mechanism, involved co-temporally in the two key processes of semen liquefaction and immune- suppression. This might be critical in protecting motile sperms following their release from semen coagulum.
Understanding KLK-mediated proteolytic events in seminal plasma can shed light not only on the physiological role of this family of enzymes, but also on some of causes of male subfertility. Accordingly, therapeutic induction of this cascade may be utilized to supplement the current clinical treatment of male subfertility. Conversely, targeted inhibition of key components of the cascade may have potential pharmaceutical utility as a novel topical contraceptive strategy.
iii
ACKNOWLEDGEMENTS
The work on this thesis has been a truly rewarding and inspiring experience, which would have been impossible without those who stood with me throughout the journey.
Foremost, I would like to express my sincere appreciation and gratitude to my supervisor,
Professor Eleftherios P. Diamandis, who patiently guided me through and helped me overcome obstacles, while deepening my passion for research with his encouragement and knowledge. His support, appreciation, and excellent advice have allowed me to advance in my research and I truly appreciate having him as a mentor throughout these years. I would also like to thank my previous supervisor, Professor Paul H. Hamel, who taught me how to think “like a scientist”.
I am also profoundly grateful for the members of my PhD committee Professors Sylvia
A. Asa, and Alex Romaschin whose advice and support helped me complete this work. I also wish to thank my PhD examination committee, Dr. Keith Jarvi and Dr. Ake
Lundwall for their time and constructive comments on my thesis draft. Very special thanks also to my collaborators, Dr. David Deperthes from MedDiscovery (Switzerland) and Dr. Juan Malm from Lund University (Sweeden) for their invaluable experience and advice.
To all members of the lab (past and present), I am very grateful for your collegial spirit and the excellent working atmosphere. I also sincerely thank my friends outside the lab,
iv
in particular Rachel, Kimi, Arash, and Sara, for their unconditional support throughout this journey and for putting up with my episodic pessimism.
Lastly, I would like to thank my family, specially my parents, Jila and Baba, my two wonderful sisters, Ariane and Azin, and my amazing aunt Sholeh, for their endless love and support. Thank you for always believing in me and helping me believing in myself.
Mom and dad, you taught me how to go through rough patches of life and by personal example, showed me to never give up. Your positive outlook on life has been a constant source of encouragement, thank you for showing me the power of perseverance.
I am truly grateful for this experience and am ending this journey realizing that “the real voyage of discovery consists not in seeking new lands but seeing with new eyes”
- Marcel Proust.
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TABLE of CONTENTS
ABSTRACT ...... iii ACKNOWLEDGEMENTS...... ivv LIST of TABLES ...... x LIST of FIGURES...... xii LIST of ABBREVIATIONS ...... xiiiii
CHAPTER 1: INTRODUCTION ...... 1
1.1. SERINE PROTEASES ...... 2 1.1.1. Classification...... 2 1.1.2. Activation Mechanism Through Proteolytic Cascades ...... 3 1.1.3. Control of Enzymatic Activity ...... 7 1.1.4. Catalytic Mechanism of Active Serine Proteases...... 8
1.2. HUMAN TISSUE KALLIKREIN-RELATED PEPTIDASES ...... 11 1.2.1. Historical Overview ...... 11 1.2.2. Gene Organization and Protein Structure...... 11 1.2.3. Phylogenetic Evolution of the Locus ...... 15 1.2.4. Substrate Specificity...... 17 1.2.5. Physiological Functions ...... 19 1.2.6. Cancer Pathobiology ...... 23 1.2.7. Nonmalignant Disorders...... 27 1.2.8. Signaling Mechanisms ...... 28 1.2.9. Proteolytic Activation Cascades...... 30 1.2.10. Regulatory Mechanisms ...... 33
1.3. HUMAN KLK14 ...... 37 1.3.1. Historical Overview ...... 37 1.3.2. Organization of KLK14 Gene and Protein Structure ...... 38 1.3.3. Substrate Specificity...... 39 1.3.4. Expression Pattern and Cellular Localization ...... 41 1.3.5. Regulatory Mechanism of Proteolytic Activity...... 41
1.4. MALE REPRODUCTIVE SYSTEM...... 43 1.4.1. Semen Composition ...... 43 1.4.2. Semen Physiology ...... 44 1.4.2.1. Sperm production and maturation...... 44 1.4.2.2. Seminal clotting and liquefaction...... 45 1.4.3. Sperm Transport in the Female Reproductive Tract ...... 46 1.4.3.1. Postmating inflammatory responses...... 47 1.4.3.2. Role of semen in maternal immune tolerance...... 47 1.4.3.3. Immune regulatory function of TGFβ in seminal plasma ...... 48
vi
1.4.4. Male Factor Subfertility ...... 51 1.4.4.1. Male infertility diagnosis ...... 51
1.5. AIM OF THE PRESENT STUDY...... 54 1.5.1. Rationale...... 54 1.5.2. Hypothesis...... 55 1.5.3. Objectives...... 55
CHAPTER 2: IDENTIFICATION OF POTENTIAL KLK14- MEDIATED CASCADE(S) ...... 56 2.1. INTRODUCTION ...... 57
2.2. EXPERIMENTAL PROCEDURES...... 58 2.2.1. Materials...... 58 2.2.2. Heptapeptide Library Screening...... 58 2.2.3. Recombinant KLK1 Production...... 60 2.2.4. Activation of ProKLK3 and ProKLK11 by KLK14 ...... 61 2.2.5. Activation of ProKLK1 by KLK14...... 62 2.2.6. N-terminal Sequencing...... 63
2.3. RESULTS...... 64 2.3.1. Heptapeptide Screening...... 64 2.3.2. Activation/Deactivation of ProKLK3 and ProKLK11...... 64 2.3.3. Cloning, Expression, and Purification of Recombinant ProKLK1 ...... 73 2.3.4. Activation of KLK1 by KLK14 ...... 73
2.4. DISCUSSION...... 78
CHAPTER 3: VALIDATION OF THE PUTATIVE KLK14-MEDIATED CASCADE IN SEMINAL PLASMA ...... 83 3.1. INTRODUCTION ...... 84
3.2. EXPERIMENTAL PROCEDURES...... 85 3.2.1. Reagents ...... 85 3.2.2. Materials...... 86 3.2.3. Enzyme-Linked Immunosorbent Assay (ELISA) ...... 86 3.2.4. Measurement of Clinical Parameters of Semen ...... 87 3.2.5. Cleavage of Sg I and II Proteins...... 87 3.2.6. Sg- Mediated Reversal of Zn2+ Inhibition ...... 88 3.2.7. Enzyme Activity Assays ...... 88 3.2.8. KLK3 Depletion From Seminal Plasma...... 89 3.2.9. Western Blotting for Identification of KLK3 Fragmentation in Seminal Plasma.90
vii
3.3. RESULUTS ...... 91 3.3.1. Clinical Association Between KLK14 Expression and Liquefaction Rate ...... 91 3.3.2. Role of KLK14 As a Seminal Liquefying Protease ...... 91 3.3.3. Cleavage of Sg Proteins by KLK14 ...... 95 3.3.4. Reversal of Zn2+ Inhibition by Sg I and II...... 95 3.3.5. Correlation Between KLK14 and the “Chymotrypsin-Like” Activity...... 98 3.3.6. Fragmentation of Seminal KLK3 by KLK14...... 100 3.3.7. Activation of Seminal KLK1 by KLK14 ...... 103
3.4. DISCUSSION...... 107
CHAPTER 4: ASSOCIATION BETWEEN SEMINAL KLKS AND MACROSCOPIC INDICATORS OF SEMEN ANALYSIS...... 112 4.1. INTRODUCTION ...... 113
4.2. MATERIALS AND METHODS...... 114 4.2.1. Clinical Samples...... 114 4.2.2. Enzyme-Linked Immunosorbent Assays (ELISA)...... 115 4.2.3. Statistical Analysis ...... 118
4.3. RESULTS...... 119 4.3.1. Distribution of KLKs Among the Four Clinical Groups...... 119 4.3.2. Association of KLKs with Liquefaction and Viscosity State...... 123 4.3.3. Association Between Semen Liquefaction State and Variables of Sperm Motility ...... 123 4.3.4. Distribution of Seminal KLKs in Asthenospermic Patients...... 127
4.4. DISCUSSION...... 131
CHAPTER 5: IDENTIFICATION OF A POTENTIAL ROLE OF MULTIPLE MEMBERS OF THE SEMINAL KLK CASCADE AS NOVEL ACTIVATORS OF THE LATENT TGFΒ1 COMPLEX IN SEMINAL PLASMA ...... 136 5.1. INTRODUCTION ...... 137
5.2. EXPERIMENTAL PROCEDURES...... 138 5.2.1. Reagents ...... 138 5.2.2. Enzymatic Activation of TGFβ1 ...... 138 5.2.3. Activation of Latent TGFβ1 by Acid Treatment...... 139 5.2.4. TGFβ1 Activity Enzyme-Linked Immunosorbent Assay (ELISA)...... 139 5.2.5. Electrophoretic Detection of Mature TGFβ1 Under Native Condition...... 141 5.2.6. In-vitro Cleavage of LAP and LTBP1...... 141 5.2.7. N-terminal Sequencing of the Newly Generated LAP and LTBP1 Fragments...141 5.2.8. Western Blotting for Identification of LAP and LTBP1 Fragmentation...... 142
viii
5.3. RESULTS...... 143 5.3.1. In-vitro Regulation of TGFβ1 Activity by KLKs ...... 143 5.3.2. KLK14 Mediated Regulation of Endogenous TGFβ1 in Seminal Plasma...... 146 5.3.3. KLK- Mediated Cleavage of LAP...... 146 5.3.4. Fragmentation of Endogenous LAP in Seminal Plasma ...... 149 5.3.5. KLK- Mediated Cleavage of LTBP1 ...... 151
5.4. DISCUSSION...... 155
CHAPTER 6: SUMMARY AND FUTURE DIRECTIONS...... 160 6.1. SUMMARY...... 161 6.1.1. Key Findings ...... 161 6.1.2. Conclusion...... 165
6.2. FUTURE DIRECTIONS ...... 168
CHAPTER 7: REFERENCES...... 171
CHAPTER 8: APPENDIX...... 192
ix
LIST of TABLES
Table Title Page
1.1 Specificity, substrates and inhibitors of human tissue kallikreins 20
1.2 Activation motifs of human tissue kallikreins 31
2.1 Relative cleavage efficiency of heptapeptides by active KLK14 65
3.1 Expression level of trypsin-like KLKs in seminal plasma 104
4.1 Descriptive statistics of patient age, semen volume, sperm counts and sperm 116 concentration in the four clinical groups 4.2 Antibodies used in the ELISA assays 117
4.3 Distribution of prostatic KLKs in seminal plasma of the four clinical groups 120
4.4 Correlation between prostatic KLKs 122
4.5 Sperm motility properties in different states of sperm liquefaction 126
4.6 KLK concentration in Normal and Asthenospermic cases 129
4.7 Correlation between prostatic KLKs and indicators of sperm motility 130
5.1 Description of KLK optimal assay buffers 140
A.1 Multiparametric models of KLK and other biomarkers in human cancers 195
x
LIST of FIGURES
Figure Title Page
1.1 Schematic representation of classic proteolytic cascades 5
1.2 Schematic presentation of serine protease catalytic mechanism 9
1.3 Schematic representation of the gene and protein of the kallikrein-related peptidase 13
1.4 Kallikrein locus conservation 16
1.5 Gene and protein characteristics of KLK14 40
2.1 Monitoring of heptapeptide (Hep) cleavage 65
2.2 Validating the cleavage specificity 67
2.3 KLK14- mediated regulation of proKLK3 activity 70
2.4 Activation of proKLK11 by KLK14 72
2.5 Activation of proKLK1 by KLK14 75
2.6 Schematic presentation of proposed kallikrein cascades in seminal plasma 76
2.7 Schematic presentation of proposed kallikrein cascades in skin 77
3.1 Clinical association between KLK14 expression and liquefaction rate and asthenospermia 93
3.2 Optical analysis of liquefaction level of semen coagulum 94
3.3 KLK14- mediated degradation of semenogelin proteins 96
3.4 Reversal of Zn2+ inhibition by semenogelin II 97
3.5 KLK3 depletion of seminal plasma 99
3.6 Regulation of total chymotrypsin activity by KLK14 101
3.7 KLK14- mediated internal cleavage of recombinant KLK3 and seminal KLK3, ex-vivo 102
3.8 KLK14- mediated activation of seminal KLK1 105
xi
List of Figures (Continue) Figure Title Page
3.9 Schematic presentation of proposed KLK cascade in seminal plasma 106
4.1 Distribution of seminal plasma KLK concentrations (ug/L) in the four clinical groups 121
4.2 KLK combination function for the prediction of liquefaction and viscosity 124
4.3 Scatter plot of KLK14 levels (µg/L) in the seminal plasma of normal and asthenospermic 128 cases 5.1 In-vitro Activation of the latent TGFβ1 145
5.2 Activation of endogenous latent TGFβ1 complex in seminal plasma 147
5.3 LAP fragmentation 148
5.4 LAP fragments in seminal plasma 150
5.5 LTBP1 fragmentation 152
5.6 Schematic presentation of the proposed functions of multiple KLKs in activation of TGFβ1 154 complex. 6.1 Schematic presentation of the proposed cascade- mediated functions of seminal KLKs 166
A1 Schematic presentation of KLK locus and their potential utility as cancer biomarkers 193
xii
LIST of ABBREVIATIONS
ACC 7-amino-4-carbamoylmethylcoumarin ACN acetonitrile ACT anti-chymotrypsin ACTP testicular acid phosphatase gene ADAMTS8 ADAM metallopeptidase with thrombospondin type 1 motif 8 ALP alkaline phosphatase AMC 7-amino-4-methylcoumarin ANF atrial natriuretic factor AP anti plasmin APMSF 4-amidino-phenyl-methane-sulfonyl fluoride ARA anthracycline resistance-associated AT antitrypsin B2R human bradykinin B2 receptor BPTI bovine pancreatic trypsin inhibitor CAG cancer-associated gene cAMP cyclic adenosine monophosphate CASA computer-assisted semen analysis CDSN corneodesmosin CNS central nervous system DHT dihydrotestosterone DSC desmocollin DSG desmoglein ECM extracellular matrix ELISA enzyme-linked immunosorbent assay EMSP enamel matrix serine protease EMT epithelial mesenchymal transition FHL four-and-a-half-LIM FN fibronectin FPLC fast protein liquid chromatography FRET fluorescence resonance energy transfer GM-CSF granulocyte-macrophage colony-stimulating factor GPCRs G-protein-coupled receptors hCAP human cathelicidin HEK human embryonic kidney hGK human glandular kallikrein HGNC HUGO Nomenclature Committee HMW high molecular weight HRE hormone response element ICSI intracytoplasmic sperm injection IGF insulin-like growth factor IGFBP IGF binding protein IL interleukin KLK kallikrein-related peptidase KNRK kirsten virus-transformed normal rat kidney
xiii
LAP latency- associated peptide (LAP LEKTI lympho-epithelial Kazal-type inhibitor LLP low density lipoprotein LMW low molecular weight LTBP latent TGFβ binding protein LTP long-term potentiation MAC membrane attack complex MAPK mitogen- activated protein kinase MBP myelin basic protein MMP matrix metalloproteinase MudPIT multidimensional protein identification technologies NS netherton syndrome OLG oligodendrocyte OMTKY3 ovomucoid third domain PAI plasminogen activator inhibitor PAP poly A polymerase PAR protease activated receptor PCI protein C inhibitor PDEF prostate-derived Ets transcription factor PKC protein kinase C pNA para-nitroanilide PSA prostate specific antigen PSGL p- selecting glycoprotein ligand PS-SCL positional scanning synthetic combinatorial library PTHrp parathyroid hormone-related peptide PVD polyvinylidene difluoride RCL Reactive center loop RP-HPLC reverse phase-high protein liquid chromatography SBzl thioBenzyl ester SC stratum corneum SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sg semenogelin SNP single nucleotide polymorphism SPINK serine protease inhibitor Kazal-1 TAP tick anticoagulant peptide TBS Tris-buffered saline TF Tissue factor TFPI tissue factor pathway inhibitor TGF tumour growth factor Th T-helper TIC total ion current TSC testicular stem cells UHN university health network uPA urokinase plasminogen activator uPAR uPA receptor UTR untranslated region
xiv
VEGF vascular endothelial growth factor VIP vasoactive intestinal peptide WHO world health organization XIC extracted ion chromatograms α2- M α2- macroglobulin
xv INTRODUCTION 1
CHAPTER 1 Introduction
Sections of this chapter were published in Clinica Chimica Acta, Molecular Oncology, and Clinical Chemistry:
Emami N and Diamandis EP. Human tissue kallikreins: a road under construction Clin Chim Acta. 2007 May;381(1):78-84.
Emami N and Diamandis EP. New insights into the functional mechanisms and clinical applications of the kallikrein- related peptidase family Mol Oncl. 2007;1:269-87.
Emami N and Diamandis EP. Utility of kallikrein-related peptidases (KLKs) as cancer biomarkers Clin Chem. 2008. In press
Copyright permissions have been granted.
INTRODUCTION 2
Proteases (also known as peptidases) are a major group of enzymes participating in multitude of physiological processes, including coagulation, apoptosis, tissue remodeling, and immune responses (1). Depending on the cleavage site, proteases are
classified as exo- and endo-peptidases(1). Endopeptidases (or proteinases) cleave their
target proteins internally, whereas exopeptidases sequentially remove amino acids from
either the N or C-terminus(1). Based on the amino acid residue present at the active site,
endopeptidases can further be grouped into four major classes of serine-, cysteine-,
aspartic-, and metallo-proteinases (1).
1.1. SERINE PROTEASES
1.1.1. Classification
Serine proteases exhibit diverse functions in digestion, coagulation, and cellular
and humoral immunity(2). Based on their amino acid sequences, 50 families of serine
proteases have been identified thus far (1). Most of these families could be further grouped into 11 clans by comparing the tertiary structure and amino acid sequence of
their catalytic residues (1). Clan PA(S) encompasses perhaps the largest number of best
known serine proteases, including trypsin and chymotrypsin (1). All proteolytic enzymes
in this clan are endopeptidases and mainly consist of β sheet in their tertiary structure (3).
The catalytic machinery in this clan relies on a catalytic triad, including the serine residue
as a nucleophilic carrier, a histidine residue as a proton donor, and an aspartate that is
essential for proper orientation of the imidazolium ring of the histidine (3). The catalytic
mechanism of serine proteases will be discussed in more detail in following sections.
The S1 family of clan PA(S) contains the largest number of sequenced proteins and different protease activities (1). Substrate specificity in this family is mainly dictated
INTRODUCTION 3 by preferences in the S1 subsite that contributes a carbonyl group to the scissile peptide bond (4). Majority of these proteases contain an N-terminal signal peptide and therefore enter the secretory pathway prior to activation (1). In addition to the soluble secretory
proteases, a number of membrane-bound proteases, including enteropeptidase, hepsin,
and matriptase, have also been reported (3). Active proteases of this subfamily are often
in their two-chain form, linked by a disulfide bridge (3).
Family S1 can further be divided into six subfamilies (1). These subfamilies were
initially thought to belong to separate families but after the discovery of linking sequences they were re-classified into a common family (1). The subfamily S1A mainly
contains animal proteases, even though proteins from Escherichia coli, Vibrio cholerae,
and Sinorhizobium have also been reported (1). The remaining subfamilies mainly contain secretory or membrane-bound bacterial proteases (1).
1.1.2. Activation Mechanism Through Proteolytic Cascades
Due to the irreversible nature of proteolytic activation, serine proteases often
remain as inactive zymogens in quiescent conditions. Activation is often achieved by
cleavage of the pro-sequence located at the N-terminal end of the protein (1). The length
of these “pro”-extensions vary, ranging from only two residues in human cathepsin G to
hundreds of amino acids found in the blood coagulation factors and complement
components (1).
Activation is often triggered by an external stimulus and mediated by highly
orchestrated cascades (Fig. 1.1)(5). These cascades can be organized into three main
consecutive phases of initiation, progression, and execution (5). Upon stimulation,
“initiator” zymogens (pro-enzymes) are self-activated by autocatalysis. Active initiators
INTRODUCTION 4 then convert downstream “progressor” proteases, which, in turn, catalyze the processing of the following “executor” zymogens. Active executers then elicit proper signals in order to repair or block adverse effects of the stimulus. Such cascades result in a rapid and highly controlled amplification of active proteases and physiologically safe proteolysis. Proteolytic cascades have extensively been examined in a large number of serine proteases and are well characterized in vital physiological processes, such as coagulation-fibrinolysis, digestive, and the complement system in both innate and acquired immunity(6-8).
The concept of proteolytic cascade was initially proposed through the extensive work on blood coagulation mechanisms about forty years ago (9;10). It is now clear that the process of coagulation consists of series of proteolytic events that are mainly localized to the surface of activated platelets. Initiation of coagulation however requires several other elements, including activated endothelial cells and leukocytes, to juxtapose coagulation zymogens and make them spatially available for their sequential activation
(11). Mechanical, chemical, or electrical injury to vessel walls is considered as the main stimulants of coagulation, functioning through P-selectin recruitment to the surface of platelet and endothelial cells (12).
INTRODUCTION 5
FIGURE 1.1. Schematic representation of classic proteolytic cascades. Cascade initiation is often induced by an external stimulus, such as injury, stress, infection. Note the three hierarchical levels of initiation, progression, and execution.
INTRODUCTION 6
P-selectin subsequently binds to its receptor, P- selecting glycoprotein ligand-1
(PSGL-1), located on membranes of neutrophils and monocytes. This binding promotes the rolling of leukocytes and platelets on the surface of activated endothelial cells (13).
Microvesicles containing primary components of the initiator protease, i.e. tissue factor-
VIIa, complexes were shown to fuse with the membrane of activated platelets, in order to make it accessible to factor X (13). Factor X is cleaved by VIIa to form factor Xa, which in turn activates factor V released from the platelet α-granulules to the surface membrane. Finally, a small amount of thrombin is formed through prothrombin conversion by factor Xa. The newly generated thrombin in turn sends positive feedback loops to activate additional platelets, factor V, and factor XI. Factor XI is bound to glycoprotein Ib/IX located on the surface of activated platelets. Activated XI sequentially activates factor IX. Activated factor IXa complexes with factor VIIIa, which is in turn activated by thrombin. In the presence of calcium, this complex activates additional factor X, which marks the initiation of the progression phase of coagulation. The large scale activation of factor Xa during the progression phase will mediate the ultimate step of thrombin formation. Factor Xa complexes with factor Va and calcium ions to form the so called “pro-thrombinase” complex. Subsequently, thrombin converts fibrinogen to fibrin. Thrombin is also able to send positive feedbacks to activate additional platelets, factor V, VIII, and XI. Finally, thrombin activates factor XIII, in order to stabilize clot formation and modulate fibrinolysis.
INTRODUCTION 7
1.1.3. Control of Enzymatic Activity
Proteolytic cascades are tightly regulated through a series of highly orchestrated feedback loops, internal cleavages, and (auto) degradations. As well, inhibitors play a major role by targeting activated proteases (5). The inhibitory mechanism of serine protease inhibitors has been characterized in detail. Proteases are shown to form transient noncovalent complexes with respective inhibitors. The complex can consequently
progress to the “inhibitory pathway” through a molecular “trap” mechanism.
Alternatively, inhibition is prevented through the “substrate” pathway whereby the protease cleaves and therefore inactivates the inhibitor (14). Inhibitors of serine proteases
can be categorized into three main groups of canonical inhibitors, serpins, and non-
canonical inhibitors (15). Canonical inhibitors, such as bovine pancreatic trypsin inhibitor
(BPTI), ovomucoid third domain (OMTKY3), or eglin, are small proteins that often form
tight, non-covalent bound with their respective enzymes in order to block enzyme’s
active site. Such reactions very closely resemble enzyme- substrate complexing (15).
Similar to canonical inhibitors, serpins form enzyme- substrate complexes with
their targets. However, unlike canonical inhibitors, the binding loop of serpins undergo conformational changes upon binding to stabilize the covalent acyl-enzyme complex
(15). Serpins are the only family of serine protease inhibitors that could complex with
non-serine enzymes, including the cysteine and aspartyl proteases (15).
Non-canonical inhibitors, such as tick anticoagulant peptide (TAP), often bind
very strongly to the active site of the protease through their N-terminal end to form a
parallel β sheet (15). These inhibitors are also able to form additional bindings, to sites
other than the active site, in order to facilitate, stabilize, and strengthen the inhibition.
INTRODUCTION 8
The aforementioned regulatory points are critical in preventing deleterious effects due to uncontrolled protease activation. Dysregulated protease activation has been implicated in several pathological conditions, such as amyloidogenesis in Alzheimer's disease, intravascular coagulation in sepsis, desquamation in various skin disorders, as well as tumour metastasis, invasion, and angiogenesis in cancer (5;16-18).
1.1.4. Catalytic Mechanism of Active Serine Proteases
Serine proteases are perhaps one of the most extensively studied group of proteolytic enzymes (1). Fundamental advances in our understanding of the mechanistic features of this family of proteases comes mainly from work carried out with chymotrypisn, initiated by Bender et al. in 1960s (19). The key player in the catalytic
mechanism in the chymotrypisn clan is the catalytic triad, consisting of three highly
conserved amino acids of histidine (His 57), serine (Ser 195) and aspartic acid (Asp 102)
(2). Located in the active site of the enzyme, these key residues play a key role in the proteolytic ability of the enzymes. Each component of this triad performs a specific and
highly coordinated process at the event of catalysis (Fig. 1.2).
According to the information acquired from the X-ray crystallography, the
geometric relation of these amino acids is crucial for proper catalytic function of the
enzyme (20). The active-site serine residue is believed to act as a nucleophile, attacking the carbonyl carbon of the scissile bond of the substrate inserted into enzyme’s active site
(19). In concert with the active-site histidine, aspartic acid function as a charge relay system, transferring the proton from the serine residue. In particular, the electron pair of the histidine nitrogen accepts the hydrogen from serine hydroxyl group.
INTRODUCTION 9
FIGURE 1.2. Schematic presentation of serine protease catalytic mechanism. Catalysis occurs in a stepwise fashion through which several intermediates are formed (Adapted from the Serine protease site of the Washington University in St.Louis, WUSTL: www.biochem.wustl.edu/~protease.
INTRODUCTION 10
Consequently, the carboxyl group of the aspartic acid has the ability to form hydrogen bonds with the histidine, augmenting the electronegativity of the electron pairs of the histidine. As a result, a covalent bond is formed between the serine and substrate to generate a tetrahedral intermediate.
The joining bond between the nitrogen and the carbon of the peptide bond is then broken mostly at the nitrogen, forming a stable acyl-enzyme intermediate. This intermediate is hydrolyzed in order to replace the N-terminus of the cleaved peptide and attacking the carbonyl carbon. This would result in formation of another tetrahedral intermediate. Finally, the C-terminus of the peptide ejected, as the bond formed between the serine and carbonyl carbon decomposes and the newly acquired hydrogen of the histidine residue transfers the proton back on the serine.
More recent evidence suggests that two additional complementary residues, namely glycine (Gly 193) and serine (Ser 195) may contribute to catalytic efficiency of serine proteases by donating backbone hydrogens for hydrogen bonding and forming a so called “oxyanion hole” (21).
INTRODUCTION 11
1.2. HUMAN TISSUE KALLIKREIN-RELATED PEPTIDASES
1.2.1. Historical Overview
Kallikrein-related peptidases (KLKs) belong to a subgroup of secreted serine
proteases within the S1 family of clan SA (22). The first member of the KLK family was
identified in 1930s, as the most abundant protease in pancreas and hence was named
tissue “kallikrein”, for pancreas (kallikreas) in Greek (23). Subsequent independent work
by Flocks, Ablin, Hara, Li and Beling, Sensabaugh, and Wang, between 1960 to the late
1970s, led to the discovery of the most well-characterized KLK, i.e. KLK3 (PSA) (23).
Subsequently, together with another novel KLK, namely KLK2 (also known as human
glandular kallikrein-1 or hGK-1), the “classic” KLK family of serine proteases was
defined (23). Further work from our laboratory and others at the end of the pre-genomic
era during the mid 1990s has eventually led to the characterization of additional twelve
novel serine protease genes, co-localized with the previously identified KLK genes (23).
According to the official nomenclature system recommended by the Kallikrein
subcommittee of HGNC (HUGO Nomenclature Committee) in 2006, to distinguish
between proteins and genes, proteins are written in standard font, e.g. KLK2, while genes
are in italics, e.g. KLK2 (24).
1.2.2. Gene Organization and Protein Structure
KLKs are encoded by a cluster of strikingly similar genes with varying length,
ranging from 4.4 to 10.5 kbp (25). The size difference is mainly attributed to intron
length, which varies significantly between the genes. Some of the common features
shared among KLKs include exon/intron organization, number and length of exonic
regions, intron phase, and conserved translational start and stop sites, as well as the
INTRODUCTION 12 catalytic triad codons (22;23). Each gene consists of 5 coding exons, separated by 4 introns with the highly conserved GT-AG splice junction pattern. Furthermore, with the exception of the “classic” KLKs that lack 5' untranslated exons, KLKs contain both 5' and
3' UTRs. The 3' UTR contains either the canonical (AATAAA) or a variant polyadenylation site distal to the stop codon (Fig. 1.3) (22).
INTRODUCTION 13
A).
B).
FIGURE 1.3. Schematic representation of the A). gene and B). protein of the kallikrein-related peptidase. KLK genes consist of 5 coding exons of similar length and 4 introns with varying size. Boxes with horizontal pattern represent the 5' and 3' UTRs. Connecting bars show intron phase. KLK proteins are expressed as pre-proenzymes. The amino-terminal pre- (signal) sequence guides the enzyme to the endoplasmic reticulum for secretion. Proenzymes are activated extracellularly upon cleavage of the pro- domain. H57, D102, and S195 are the amino acids of the catalytic triad.
INTRODUCTION 14
KLK proteins are secreted serine endoproteases, expressed as single chain preproenzymes of approximately 30-40 kDa (Fig. 1.3) (25). The signal (pre-) sequence is
16-30 amino acids in length and is cleaved from the N-terminus of the protein prior to secretion (23). Enzyme activation may subsequently occur through limited proteolysis targeted to the peptide bond between basic and hydrophobic residues of the “pro”- sequence (26). Characteristic to serine proteases, KLKs contain a catalytic serine residue at their active site cleft. Along with the active serine, histidine and aspartic acid residues of the catalytic triad, serve as a charge relay system (25;26).
KLKs share a high level of amino acid identity in areas flanking the catalytic triad(25). The overall sequence similarity, however, is estimated at a lower level (40% to
80%) with highest sequence similarity between the “classic” KLKs (25).
So far, the 3D structure of mature KLK1, KLK4, and both mature and pro-KLK6 have been determined by X-ray crystallography (26-29). As a subgroup of the trypsin/chymotrypsin-like serine proteases, these KLKs are folded into two hydrophobically interacting domains of six-stranded β-barrels and an α-helix. The catalytic triad is located at the interface between the two domains, as a shallow depression on the frontal surface (26;27;29).
KLK1 contains an additional “kallikrein loop”, unique to the “classic” KLKs
(28). The loop consists of 11 amino acids, inserted at position 95 (28). Given its close proximity to the active site, the kallikrein loop is believed to affect the substrate accessibility of the enzyme (28). Substrate/inhibitor binding may also be determined by diverse external loops surrounding the substrate binding sites (29).
INTRODUCTION 15
1.2.3. Phylogenetic Evolution of the Locus
The KLK locus resides on the long arm of chromosome 19, at position q13.4 and is confounded centromerically by the testicular acid phosphatase gene (ACTP) and telomerically by the cancer-associated gene (CAG) and Siglec-9, a member of the sialic acid-binding Ig-like lectin family (22;30;31). In human, KLK are organized as the largest uninterrupted tandem array of protease genes and are transcribed from telomere to centromere, with the exception of KLK2 and KLK3 (Fig. 1.4) (25).
Phylogenetic and comparative analyses of the KLK locus have revealed a significant level of locus similarity among mammals, suggesting a conserved function(s) of the encoded proteins (32;33). Experimental and in-silico identification of mammalian
KLKs in human, rat, mouse, pig, dog, chimpanzee, and opossum, as well as comparative studies of the genome of horse and cow have revealed a polyphyletic nature of the gene family (32-34). Further phylogenetic studies have suggested five main subfamilies with shared recent ancestry, namely KLK4, 5, 14; KLK9, 11, 15; KLK10, 12; KLK6, 13; and
KLK8, 1, 2, 3 (33). No KLK was found in the nonmammalian species examined thus far
(33).
Bayesian phylogenetic analyses of the KLK locus of the genome of human, chimpanzee, mouse, rat, dog, pig, and opossum indicate that these species carry at least one copy of the KLK5-15 (33). Interestingly, “classic” KLKs exhibit the most variability in the number of gene copies, with the highest number of duplications in rodents (33).
Given that the number of gene copies are similar in marsupial species, the majority of duplication events probably date back to 125-175 million years ago, prior to the marsupial-placental divergence (33).
INTRODUCTION 16
FIGURE 1.4. Kallikrein locus conservation. Arrowheads indicate the approximate location of genes and their transcription direction. Light gray, “classical” kallikreins, gray, newly discovered kallikreins, Black, pseudogenes, Red, non-kallikreins. Figure is not to scale [Modified from ref.(33)].
INTRODUCTION 17
Despite major progress in understanding of the phylogenetic changes of the KLK family, evolutionary processes of KLK4/KLK5 has mainly remained a mystery. KLK4 is reportedly missing in the mono-delphine genome (33). However, whether the gene was deleted or was duplicated post marsupial lineage divergence is still not clear. Given that
KLK4 is highly varies among other mammalian species supports the latter hypothesis
(33). Lastly, the phylogenetic tree constructed from both individual genes and concatenated KLKs suggest a tandem duplication mechanism of sister taxa, including
KLK9/KLK11 and KLK10/KLK12 (33).
The predicted number of clades is expected to decline as the genome of more primitive mammals becomes available. The rapidly accumulating phylogenetic data are expected to provide new clues on the biological significance of the family.
1.2.4. Substrate Specificity
Based on their substrate binding pocket, KLKs are broadly grouped into two clans of chymotrypsin-like and trypsin-like serine proteases(23). Experimental and in-silico analyses indicate that the majority of KLKs, namely KLK1, 2, 4-6, 8, 10-15, contain an asparatic or glutamic acid residue at the base of their substrate pocket, enabling them to cleave peptide bonds following a positively-charged amino acid residue (23). The remaining KLKs, i.e. KLK3, 7, and 9, contain a hydrophobic pocket mainly suited for cleavage of substrate scissile bonds with bulky hydrophobic amino acids such as phenylalanine, tryptophan and tyrosine (23).
Substrate specificities of a large number of KLKs have been determined experimentally, using diverse techniques such as phage display, combinatorial libraries, fluorescence resonance energy transfer (FRET) peptide libraries, and kinetic assays (35-
INTRODUCTION 18
38). Substrate selection through the phage display is carried out, using a library of random nucleotide sequences coding all possible combinations of amino acids. These sequences are expressed at the phage surface. Recombinant phages are then fused to a ligand and immobilized on an affinity support through a receptor. Phages expressing desired substrates are released by proteolysis with the protease of interest. Selectivity is improved by multiple rounds of selection. Finally, fragments cleaved by the protease are identified by sequencing phage DNA (35). So far, phage display technology has been utilized in substrate recognition of KLK2 and 14 (39;40). KLK14 exhibits both trypsin and chymotrypsin-like activity, which has further been confirmed using kinetic assays
(40;41). Substrate specificity of KLK14 will be discussed in more detail in the following sections.
Alternatively, positional scanning synthetic combinatorial libraries (PS-SCLs) can be employed to determine substrate recognition (42). The newly modified PS-SCL screening approach, using ACC (7-amino-4-carbamoyl methylcoumarin) as the fluorogenic leaving group, has emerged as an alternative approach for rapid substrate profiling (37). In this approach, a library comprised of 4 sublibraries of fixed P1-4 positions, each containing the twenty canonical amino acids, is constructed. The three remaining positions of each sublibrary contain an equimolar mixture of amino acids (43).
This approach has been used to verify substrate preference of KLK3-7 and 10-11 (43).
Interestingly, KLK10 and 11 were shown to have a dual chymotrypsin- and trypsin- like substrate specificities (43). Similarly, a library of FRET peptides has been utilized in
KLK6 substrate identification (38).
INTRODUCTION 19
Lastly, potential endogenous substrates of several KLKs have been identified, using fluorogenic or colourimetric conjugated, or full-length substrates (Table 1.1).
1.2.5. Physiological Functions
KLKs are expressed in diverse cell populations and have been implicated in a wide range of physiological processes. Due to their early discovery, functional roles of the “classical” KLKs have long been studied. KLK1 is expressed in a large number of tissues, including kidney, blood vessels, central nervous system, pancreas, gut, salivary and sweat glands, spleen, adrenal and neutrophils, suggesting a paracrine nature of the enzyme (44). KLK1 has also has been detected in plasma, possibly originating from exocrine glands (44). KLK1 primarily functions through the release of kallidin (KD) mainly from the low molecular weight kininogen (LK) (44). The kinin-mediated signaling pathway of KLK1 has been implicated in a number of processes, including regulation of blood pressure, smooth muscle contraction, neutrophil chemotaxis, pain induction, vascular permeability, electrolyte balance, and inflammation (22). Additional functions associated with KLK1 include processing of growth factors and peptide hormones, increased nitric oxide formation, and reduced oxidative stress (22;45). Recent evidence suggest that KLK1 may also function independent of kininogens (46).
The remaining “classic” KLKs, KLK2 and 3, have extensively been examined due to their restricted expression mainly in prostate and seminal plasma. Physiological function of KLKs in seminal plasma will be discussed in detail later on.
INTRODUCTION 20
Table 1.1. Specificity, substrates and inhibitors of human tissue kallikreins
Kallikrein S1 amino Substrate Candidate physiologic substrate Candidate physiologic inhibitors acid specificity KLK1 Asp Trypsin-like LMW kininogen, pre ANF, pro-insulin, LLP, Kallistatin, PCI, α1AT, placental Prerenin, VIP, procollagenase, bikunin (22) angiotensinogen, B2R (22), Pro-MMP2, 9, IGFBP3 (47)
KLK2 Asp Trypsin-like Seminogelin I/II, fibronectin, pro-uPA PCI, PI-6, PAI-1, ATIII, α2M (22) (22;48), IGFBP 2, 3, 4, 5(48),ADAMTS8, collagen IX-α chain (39)
KLK3 Ser Chymotrypsin- Seminogelin I/II, fibronectin, laminin, ACT, , α2M, PCI, α1AT, ATIII (22) like lysozyme, plasminogen, TGF-β, PTHrp (22), IGFP3, 4 (48)
KLK4 Asp Trypsin-like Pro-uPA, PAP(22), enamelin(49) α2M, α2AT, α2AP(50)
KLK5 Asp Trypsin-like Collagens type I, II, III, IV, fibronectin, α2M, α2AP, ATIII(22), LEKTI (53) laminin, plasminogen, LMW kininogen, fibrinogen(51), hCAP18(52)
KLK6 Asp Trypsin-like Fibrinogen, fibronectin, laminin, collagen ATIII, α2AP, α1AT, ACT (22) types I and IV, APP, plasminogen(22), MBP, ionotropic glutamate receptor(38)
KLK7 Asn Chymotrypsin- IL-1β, corneodesmosin (22), hCAP18(52), LEKI (53), PCI, α1AT, α1 ACT, like fibrinogen(47) kallistatin (54)
KLK8 Asp Trypsin-like fibronectin, gelatin, collagen type IV, antipain, chymostatin, leupeptin (55) fibrinogen, and HMW- kininogen,
KLK9 Gly Chymotrypsin- plasminogen activator (55), (56) like
KLK10 Asp Ambivalent
KLK11 Asp Ambinalent PCI (54), APMSF, Aprotinin (57) KLK12 Asp Trypsin-like α2 AP, PCI (54),α2 AT (submitted for publication) KLK13 Asp Trypsin-like ECM, plasminogen (22) α2M, α2AP, ACT (22) hj KLK14 Asp Ambivalent collagens I-IV, fibronectin, laminin, α1-AT, α2- AP, AT III and α1- ACT kininogen, fibrinogen, plasminogen, (59) vitronectin and IGFBP 2, 3 (58), matrilin4 (47) KLK15 Glu Trypsin-like Please see “abbreviation” for full names of proteins.
INTRODUCTION 21
Furthermore, recent studies provide compelling evidence that KLKs may play an essential role in the normal physiology of skin. KLK5 and 7 were originally isolated and cloned from the stratum corneum (SC), the outermost layer of skin (60;61). Subsequent substrate analysis suggested that these KLKs might be involved in skin desquamation through processing of main adhesive proteins of the extracellular corneodesmosomes, i.e. corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) (62). KLK5 was shown to cleave all three components, while KLK7 was able to digest only CDSN and DSC1 (62). Further immunohistochemical studies revealed the subcellular localization of KLK7 in lamellar bodies in the stratum granulosum and its subsequent transport to the extracellular space of SC, supporting the proposed role of KLK7 in desquamation (63). Additional in-vitro studies suggested an activation mechanism of
KLK7 through a proteolytic cascade, involving KLK5, 7, and 14 (64). In addition to
KLK5 and 7, varying levels of KLKs 1, 6, 8, 10,11, and 13 have been reported in SC
(65;66). KLK1, 5, 6, and 14 are believed to be involved in skin desquamation through processing of DSG1 (66). In particular, KLK14 has been suggested to play a major role in skin remodeling as it contributes to approximately half of the total trypsin-like proteolytic activity in the SC layer (67). Similarly, based on the reported features of
KLK8 knockout mouse, KLK8 may play an overlapping function in skin desquamation through processing of DSG1 and CDSN (68).
Recent data has shown an additional antimicrobial function of KLKs in skin through the regulation of cathelicidin peptides (52). KLK5 and 7 were found to efficiently cleave cathelicidin precursor (hCAP18) to its mature form (LL-37), in-vitro
(52). Moreover, mice lacking the serine protease inhibitor LEKTI exhibit an increased
INTRODUCTION 22 antimicrobial activity in skin, further supporting the hypothesized function of KLKs in skin immune defense (52).
Several reports suggest a possible role of KLKs in the processing of hormones.
For instance, a large number of KLKs (KLK5-8 and 10-14) are reportedly expressed in the pituitary gland, some of which co-localize with the human growth hormone (hGH)
(69). With the exception of KLK10-12, these KLKs were shown to cleave hGH, in-vitro
(69). Similarly, various immunohistochemical reports suggest that KLK1, 6, 10, and 13 are strongly expressed in the islets of Langerhans and may regulate prohormone activation of insulin, glucagon, somatostatin, and pancreatic polypeptide (70-73).
Furthermore, accumulating data suggest a potential role of KLKs in the central nervous system (CNS). So far, the main focus has been on KLK6 and 8, as they show a distinct expression pattern in the CNS of adult human (74). KLK6 was reportedly expressed in the peripheral nerves, choroid plexus epithelium, and some neuroendocrine cells of the CNS (70). Similarly, KLK8 is preferentially expressed in adult CNS, particularly in the brain (74;75). Despite the convincing expression data in human, most of our current knowledge of KLK physiology in the CNS comes from the work done on rodents. It is reasoned that since these KLKs exhibit a high level of similarity (>70%) to their rodent orthologs, the function of these proteins is more likely conserved (22;76).
There is accumulating data that rodent KLK6 and 8 are critical in neural and brain development. For instance, KLK8-/- mice exhibit a severe loss of long-term potentiation
(LTP), required for the hippocampus-associated memory formation (77).
KLK8 is believed to be involved in long term potentiation (LTP) by modifying the
morphology of excitatory synapses by changing their adhesiveness (77). Analogous to
INTRODUCTION 23
KLK8, the mouse ortholog of KLK6 has been suggested to play a major role in CNS development through the maintenance of myelination in oligodendrocytes (OLGs) (78).
Similarly, the rat ortholog of KLK6 has been implicated in the regulation of CNS demyelination (38).
Lastly, a putative function for KLK4 has recently been proposed, based on the expression profile, mutational pattern, and substrate specificity of the protein. KLK4 was originally purified and cloned from porcine enamel extract and was designated as enamel matrix serine protease 1 (EMSP1) (79). Subsequent mutational analysis of individuals with amelogenesis imperfecta has revealed a mutation in the KLK4 gene, suggesting a possible role of KLK4 in enamel formation (80-84). Using purified porcine proteins, it was further shown that KLK4 cleaves the 32-kDa fragment of enamelin, normally accumulating in the deeper layers of enamel (49).
1.2.6. Cancer Pathobiology
Accumulating evidence indicates that the KLK family is dysregulated in cancer.
Notably, KLKs exhibit a differential expression pattern and confer a coordinated pattern of up- or down- regulation (47). Given their distinct expression profile in various malignancies, particularly in endocrine-related carcinomas, the KLK family was shown to represent a rich source of tumour biomarkers (Appendix, Fig. A1).
Molecular biomarkers of cancer may be used for screening, diagnosis, prognosis, tumour staging, monitoring of pharmacological response to a therapeutic intervention, and establishing tumour recurrence or remission (85). The interest in KLKs as cancer biomarkers dates back only 28 years ago to Papsidero’s attempt to measure PSA/KLK3 quantitatively in serum and subsequent clinical work on the potential use of the protein as
INTRODUCTION 24 a marker of prostate cancer (86). Since then, PSA /KLK3 has gained tremendous popularity as a prostate cancer biomarker. Given the structural similarity between
PSA/KLK3 and other KLKs, the potential role of the remaining members of the family as cancer biomarkers has been widely investigated in recent years. In addition to KLK3,
KLK2, 5, 11, 14, and 15 might function as complementary biomarkers in diagnosis/prognosis of prostate cancer (47;87;88). Similarly, KLK3, 5, 7, 9, 14, 15 represent potential biomarkers for breast cancer (87;89-93). Finally, KLK5-11, 13, and
14 have been suggested as tumour biomarkers for ovarian cancer (90;91;94-99). While primarily known for their biomarker value in prostate, ovarian, and breast cancers, more recent data suggest analogous roles of KLKs in several other cancers, including gastrointestinal, head and neck, lung and brain malignancies (100;101). Current attempts have primarily focused on exploring biomarker panels to increase the accuracy of prognosis, prediction of therapy, or diagnosis. To date, multiparametric KLK panels have been proposed for prostate (102-104), ovarian (105;106) and lung cancers (107)
(Appendix, TableA1).
Despite significant progress in understanding the biomarker utility of the KLK family, their (patho)physiology in cancer remains insufficiently understood. Emerging evidence indicates a possible role of the KLK family in diverse cancer-related processes, including tumour growth, angiogenesis, invasion, and metastasis.
KLK- mediated tumour growth is believed to be modulated mainly through insulin-like growth factors (IGFs) (47). For instance, KLK2, and 3 were shown to cleave a number of IGFBPs and as a result, may indirectly be involved in tumour growth (48).
Based on the substrate specificity of the remaining KLKs, KLK4, 5, and 14 are also
INTRODUCTION 25 suggested as potential upstream regulators of IGFBPs (51;108;109). Conversely, KLK3 and 10 have been implicated in tumour growth suppression. KLK3 was shown to induce the expression of putative tumour-suppressor genes, including IFN-δ, and suppress tumor growth promoters, such as uPA, VEGF, and Pim-1 oncogene, in the PC-3M prostate cancer cell line treated with free KLK3 purified from seminal plasma (110). However, despite the evidence in favour of the tumour-inhibiting role of these KLKs, their pathological function in-vivo is still controversial. For instance, one study reports that
KLK3 has no inhibitory effect on the growth of prostate cancer cell lines PC3, DU145, stably expressing pro-KLK3 (111).
In addition, KLKs are believed to be directly involved in the process of endothelial cell invasion and migration by processing ECM components. KLKs may also mediate ECM remodeling indirectly through the MMPs, uPA, and kinin signaling pathways. For instance, KLK1 activates pro-MMP1, 2, and 9 (112-114). Furthermore,
KLK2 , 4, and 12 signal through the uPA system, which results in plasmin formation
(115-118). Plasmin, in turn, degrades a large number of ECM proteins, including fibronectin, laminin, proteoglycans, and fibrin, and activates latent collagenases (119).
Likewise, KLK1 and 12 are expressed by endothelial cells and are believed to function through the kinin signaling pathway (116;120). Active kinin promotes angiogenesis by upregulation of bFGF or stimulation of VEGF formation (121). In contrast, certain KLKs could suppress angiogenesis. For example, KLK3, either purified from the seminal plasma or expressed recombinantly, was shown to prevent angiogenesis in-vitro
(122;123). The anti-angiogenic effect of recombinant KLK3 was further demonstrated in- vivo, using matrigel plug assay in wild type mice (123). Interestingly, the antagonistic
INTRODUCTION 26 function of KLK3 was independent of its enzymatic activity (123). KLK3 is believed to prevent vasculature formation through angiostatin-like components, potent inhibitors of endothelial cell proliferation and angiogenesis (124). In addition to KLK3, in-vitro data indicate that KLK5, 6, and 13 can potentially generate angiostatin-like fragments from plasminogen (51;120;125;126).
Furthermore, KLK3 and 4 have been reported to be involved in phenotypical changes that could be indicative of EMT (127). Stable expression of these KLKs in the prostate cell line PC-3 resulted in an increase in cell invasiveness (127). Transfected cells reportedly acquired mesenchymal characteristics and lost certain morphological features unique to epithelial cells (127). Predominantly, a significant loss of E-cadherin, a member of the calcium-dependent cell-cell adhesion molecules, and expression of the mesenchymal molecule vimentin were observed (127). Accordingly, the observation that suppressing KLK3 attenuates invasion in the LNCaP prostate cancer cells (128;129) is consistent with the proposed function of KLK3 in EMT. However, a reduced number of surface lung metastases was found in mice treated with KLK3, suggesting that KLK3 inhibits tumour metastasis (122). Whether the metastatic role of KLK3 is cell-specific or is counterbalanced by its anti-angiogenic effect in-vivo needs to be further investigated.
Lastly, recent findings indicate a possible role of several KLKs in bone metastasis. Bone metastasis in cancer is broadly divided in two categories, osteoclastic and osteoblastic, based on the type of activated precursor cells. Even though these classes are non-exclusive, one often predominates the other, depending on the neoplastic origin of the tumour (130). In addition to their proposed function in EMT, KLKs have been implicated in metastatic dissemination through ECM remodeling. Several lines of
INTRODUCTION 27 evidence indicate that KLK3 can induce osteoblastic proliferation and osteoclast apoptosis in-vitro and in-vivo (131;132). Although the functional mechanism of KLK3- induced bone metastasis is not fully understood, an autonomous function independent of tumour growth factors has been proposed (132). However there are several reports indicating a possible signaling through latent transforming growth factor (TGF) β or other cell surface receptors (132;133).
1.2.7. Nonmalignant Disorders
As discussed previously, KLKs play an important role in SC desquamation and are critical in the maintenance of skin barrier function. Desquamation is a complex biological event, exquisitely regulated through a series of biological checks and balances.
Imbalances in the proteolytic activity of KLKs, either as a result of gene over-expression or dysregulated activity, is considered as one of the main etiological factors in a number of skin disorders, including chronic itchy dermatitis, peeling skin syndrome, psoriasis, atopic dermatitis, and Netherton syndrome (134-138).
Clinical studies indicate that the expression of multiple KLKs is significantly up- regulated in psoriasis, atopic dermatitis, peeling skin syndrome type-B, and chronic lesions of atopic dermatitis (134;136;137). Furthermore, mutational analyses in patients with Netherton syndrome, an autosomal recessive skin disorder, have identified several frame shifts and non-sense mutations in the SPINK5 gene encoding for LEKTI (139-
141). Such genetic defects lead to truncation of the protein and loss of inhibitory domains
(140;141). As mentioned previously, LEKTI is a serine protease inhibitor shown to repress the proteolytic activity of several KLKs, including KLK5, 6, 7, 13, and 14
(66;142). As expected, a reduced level of LEKTI domains and uninhibited serine protease
INTRODUCTION 28 activity of KLKs have been observed in the SC of NS patients, as well as the disease model, namely spink5-/- mice (17;135;139;143).
According to clinical data and their putative physiological functions, several
KLKs have been implicated in a number of other disorders, including oral and
maxillofacial and neurodegenerative disorders. For instance, the expression of KLK6, 7,
and 10 are reportedly altered in patients with Alzheimer's disease and frontotemporal
dementia, which may have some utility as diagnostic biomarkers (74;144). Lastly, aberrant KLK-kinin signaling and their role in a wide range of pathological processes, including inflammation, hypertension, and renal diseases have extensively been investigated (44;145;146).
1.2.8. Signaling Mechanisms
Emerging evidence suggests that KLKs function partly through cross-talk with other signal transduction pathways. Signaling through active kinins, uPA, protease
activated receptors (PARs), and MMPs have so far been examined (147). KLK signaling
through kinins is one of the most well-characterized signaling pathways studied thus far.
KLK1, 2, and more recently 12, were shown to release active kinins (Lys-bradykinin or
kallidin) from the kininogens, in particular the low molecular weight kininogens (LK)
(116;148). Subsequently, active kallidin mediates signaling mainly through two types of
G-protein-coupled receptors (GPCRs), designated as B1 and B2 (44). The binding of kinin peptides to their respective receptors activates a number of downstream targets such as nitric oxide (NO), cGMP, prostacyclin and cAMP, which in turn induce a wide range of biological processes involved in angiogenesis, vasodilatation, smooth muscle contraction/relaxation, inflammation and pain (149).
INTRODUCTION 29
In addition to the kinin system, certain KLKs, e.g. KLK2 and 4, can cross-talk with the uPA-uPAR signaling pathway. KLK2 was shown to cleave and activate the single chain uPA at Lys(158) (118). Further studies indicate an alternative route through
complexing and inactivation of PAI-1, the main inhibitor of uPA in tissues (150).
Similarly, as mentioned previously, active chimeric KLK4 was found to activate uPA, in-
vitro (115). Plasminogen activation through the uPA/uPAR signaling has been implicated
in a broad spectrum of biological effects, including cleavage of various ECM components
and MMP activation. MMP activation has also been suggested to occur directly through
several KLKs (47).
Lastly, emerging evidence suggests that KLKs can activate and signal through
several members of the PAR family (151). PARs are members of the seven
transmembrane GPCR superfamily and are activated by serine proteases (152).
Activation is achieved via cleavage of a portion of the extracellular N-terminus of the
receptor, resulting in formation of a tethered ligand and activation of the cleaved receptor
(152). The PAR family functions through the recruitment of several different
heterotrimeric G proteins. The activation of Gq results in Ca2+ mobilization and PKC
activation through its α-subunit (152), whereas signaling through Gi can suppress cAMP
formation through adenylyl cyclase suppression (152). Alternatively, the βγ-complex of
Gi can induce a number of tyrosine kinases and subsequent activation of the MAP kinases
(152).
In a recent study, KLK5, 6, and 14 were shown to cleave the activation domain of
PARs 1, 2, and 4, in-vitro (151). KLK14-mediated cleavage of PAR2 was further
confirmed in rat PAR2-expressing KNRK rat kidney cells (151). As mentioned above,
INTRODUCTION 30
PARs have been implicated in Ca2+ mobilization. The Ca2+- mediated signaling was
shown to be modulated in KNRK cells by all three KLKs, with the highest level in
KLK14 (151). A similar result was observed in the HEK human embryonic kidney cells
transfected with PAR1, PAR2, or both (151).
As well, KLK14 was found to have a preference towards PAR2, as determined by
pre-desensitization of receptors (151). PAR1 and 2 activation was further confirmed in-
vivo, as a distinct rat/mouse aorta ring relaxation was observed upon KLK treatment
(151). Additional data indicates a possible negative regulatory feedback system through
which KLK14 deactivates PAR1. Lastly, analogous to PAR2, PAR4 was found to be
activated by KLK4 in the rat platelet cells lacking other PARs and PAR4- transfected
HEK cells (151).
1.2.9. Proteolytic Activation Cascades
There is accumulating evidence suggesting that KLKs exert their physiological
function through highly regulated proteolytic cascades. All KLKs, with the exception of
KLK4, contain a pro-peptide with lysine or arginine in their C-termini, suggesting their
activation by trypsin-like proteases (Table 1.2).
INTRODUCTION 31
Table 1.2. Activation motifs of human tissue kallikreins
1 KLK Activation motif proKLK1 I-Q-S-R↓I-V-G
proKLK2 I-Q-S-R↓I-V-G
proKLK3 I-L-S-R↓I-V-G proKLK4 S-C-S-Q↓I-I-N
proKLK5 S-S-S-R↓I-I-N
proKLK6 E-Q-N-K↓L-V-H
proKLK7 Q-G-D-K↓I-I-D
proKLK8 Q-E-D-K↓V-L-G
proKLK9 A-D-T-R↓A-I-G
proKLK10 N-D-T-R↓L-D-P
proKLK11 G-E-T-R↓I-I-K
proKLK12 A-T-P-K↓I-F-N
proKLK13 E-S-S-K↓V-L-N
proKLK14 D-E-N-K↓I-I-G
proKLK15 D-G-D-K↓L-L-E 1 Activation sites are shown by arrows. Note that, with the exception of KLK4, all KLKs are activated upon cleavage after arginine or lysine amino acid residues.
INTRODUCTION 32
However, as mentioned previously, some of the KLKs are chymotrypsin-like and thus require other trypsin-like proteases for their activation. These data are suggestive of a network consisting of multiple KLKs, being linearly activated. Moreover, the majority of KLKs exhibit common regulatory mechanisms (through steroids) and dysregulation patterns in various pathological conditions, which further supports the proposed cascade- mediated activation mechanism (153).
KLK5, 14, and 7 are postulated to participate in a proteolytic cascade in the skin
(64). KLK5 and 7 were originally isolated and cloned from the SC layer of skin (60;61).
In-vitro data suggest that KLK5 autoactivates and activates KLK7 and 14. In turn, activated KLK14 is believed to send positive feedbacks to amplify KLK5 activation.
Actived KLK5, 7 and 14 function in skin desquamation through degradation of corneodesmosomal proteins, i.e. DSG1, DSC1, and CDSN. KLK5 was shown to cleave all three components, whereas KLK7 and KLK14 were able to digest only CDSN and
DSG1, respectively (62;66). Over-desquamation in a number of skin disorders, such as
Netherton syndrome, has mainly been attributed to dysregulted proteolytic activity of these KLKs (53;136). As well, KLK5 and 7 posses antimicrobial function in skin, presumably through a cascade-mediated cleavage of the cathelicidin precursor, hCAP18, to its antimicrobial active form (LL-37) (52).
Additional evidence supporting proteolytic cascades of KLKs comes from the work done with KLK2, 3, and 5 in seminal plasma and in-vitro. KLK5 has been shown to autoactivate, and in turn, activate pro-KLK3(154). Activated KLK3 is consequently inactivated by KLK5, through a series of internal cleavages (154). Similarly, even though debatable (155), active KLK2 has been reported to cleave and activate pro-KLK3 in-
INTRODUCTION 33 vitro(156;157). Activated KLK2 and 3 may contribute to seminal clot liquefaction through hydrolysis of seminal vesicle proteins, i.e. semenogelins (Sg) I and II, and fibronectin (FN) (158). Semen liquefaction is under a tight regulatory control by a number of endogenous inhibitors such PCI, as well as inhibitory Zn2+ (159-161). Several
other KLKs, including KLK1, 11, and 14 are known to be expressed in varying levels in
seminal plasma (57;90;162), and may be involved in a common activation pathway.
1.2.10. Regulatory Mechanisms
Accumulating evidence indicates that the majority of KLKs are transcriptionally
regulated through steroid hormone. KLK expression was shown to be significantly
induced in steroid-treated cell lines both at the mRNA and protein levels (23).
Subsequent in-silico and deletion analysis identified a number of hormone response
elements (HREs) distal to transcriptional start sites of several KLKs. For example, three
androgen response elements have been identified in the KLK3 promoter. Two of these
regulatory regions, identified at -170 to -400 bp of KLK3 promoter regions, were shown
to act cooperatively (163). A SNP (single nucleotide polymorphism) located at the first
regulatory region was further shown to associate with receptor binding and KLK3
expression (164). In addition, a more potent enhancer, located at ~ -4,000 bp, was
reported and verified using DNAseI-hypersensitivity assay (165;166). Similarly, two
androgen regulatory regions, at position -170 bp and -3,000 bp, have been determined
experimentally in KLK2 (167;168). Further studies suggest that additional regulatory
factors are indirectly involved in the transcriptional regulation of these KLKs. For
instance, a Fos-containing protein complex was found to bind distal to the AREs of KLK2
and KLK3 promoters and regulate androgen-mediated gene expression (169). As well, a
INTRODUCTION 34 number of co-regulatory factors, e.g.SRC1and 3, ARA (anthracycline resistance- associated) 24 and 54, FHL (four-and-a-half-LIM )2, and PDEF (prostate-derived Ets transcription factor) have been identified in varying amount in several breast cancer cell lines (170). It has been postulated that these factors modulate the expression of KLK2 and 3, in cooperation with androgen receptors (170). In addition, androgen-mediated gene expression in KLK3 was found to significantly be potentiated, through a novel regulatory element (171). In contrast, a negative cis-acting regulatory region, recruiting both the p65 component of the NF-κB and androgen receptor, was identified in the promoter region of
KLK3 (172). Similarly, androgen-mediated KLK3 expression was found to be significantly reduced in prostate cell lines with constitutively active Ras/MAPK
(mitogen- activated protein kinase) pathway (173), suggesting the regulatory function of receptor phosphorylation in KLK expression.
Recent reports demonstrated a synergistic hormonal regulation in KLKs, suggesting a control mechanism through single locus regions (174). For instance KLK10,
11, 13, and 14 were found to be coordinately regulated by dihydrotestosterone (DHT) and norgestrel in several breast cancer cells (174). Interestingly, none of these genes contain characterized HREs, suggesting an indirect function of steroid hormones as trans-acting transcriptional regulators of KLK expression (174;175).
Alternatively, KLK expression can be regulated through epigenetic factors, in particular DNA methylation. For instance, KLK10 downregulation has been associated with the hypermethylation of CpG islands in breast cancer and lymphoblastic leukemia
(176;177). A similar regulatory mechanism has been reported for the KLK6 gene(178).
INTRODUCTION 35
Prost- translationally, the proteolytic activity of KLKs is believed to be regulated at the level of zymogen activation and/or, later on, through endogenous or small molecule inhibitors. Several in-vitro studies suggest that KLK activation is regulated through various regulatory feedback loops. For instance, KLK5 activation was shown to be positively regulated by active KLK14, while negatively controlled by active KLK 3
(64;154). In addition, several reports indicate a possible inactivation mechanism in
KLK2, 6, 7, 13, and 14 through internal cleavages and subsequent degradation
(60;126;179). Degradation may be autolytic or mediated through other proteases.
Furthermore, divalent ions such as zinc have been shown to reversibly inhibit certain
KLKs, including KLK2, 3, and 5 (47;51). These control mechanisms are of essential importance in-vivo, as they assure an adequate physiological response.
Alternatively, protease activity can be regulated through endogenous inhibitors.
The inhibitory mechanism of the serine protease inhibitors has been characterized in detail. Proteases are shown to form transient noncovalent complexes with respective inhibitors. The complex can consequently progress to the “inhibitory pathway” through a molecular “trap” mechanism. Alternatively, inhibition is prevented through the “substrate pathway” whereby the protease cleaves and therefore inactivates the inhibitor (14).
A large number of potential endogenous KLK inhibitors have been identified in- vitro (Table 1.1). Complex formation of some of these inhibitors has been proven in-vivo.
For instance, the majority of serum KLK3 (70%- 90%) was found to complex with ACT, a member of the serpin family (180).
Most of the recognized inhibitors exhibit a relatively low level of specificity. For instance, PCI was found to efficiently inhibit KLK1, 2, 3, 5, 7, 8, 11, 13, and 14 (54;181).
INTRODUCTION 36
The most specific inhibitor identified so far is kallistatin, an inhibitor of KLK1 and
7(54;182). An amino acid residue, Phe(387), has been shown to be essential in the specificity of the inhibitory function of the protein by retaining the hydrophobicity required for the optimal interaction with KLK1 (183). Further structural analysis has revealed a secondary binding site between the H helix and the C2 sheet possibly facilitating complex formation (183).
Alternatively, inhibition specificity can be achieved in-vivo by directing the inhibitor to the location of its protease target. This mechanism was demonstrated for the
KLK inhibitor LEKTI. Immunohistochemical analysis has shown a temporal compartmentalization of LEKTI and its target KLKs, e.g. KLK5 and 7, in the lamellar granule of normal skin. It has been suggested that in normal physiological state, LEKTI is transported earlier to prevent unwanted proteolytic activity of KLKs and a premature corneocyte desquamation (184). LEKTI contains 15 inhibitory domains, two of which, namely domains 2 and 5, contain three sulfide bonds characteristic of Kazal-type domains (185). Despite of the difference in sulfide bonds, domain 6 was shown to consist of two helices and a β-hairpin, found in Kazal-type domains (186). Further kinetic analysis of domains 1-6, 6-9, 9-12, and 12-15 showed a strong inhibition of KLK5, 6, 13, and 14 by the three former regions (66). As well, domain 6 of LEKTI was found to inhibit KLK7 (142).
INTRODUCTION 37
1.3. HUMAN KLK14
1.3.1. Historical Overview
KLK14 also known as KLK-L6, was discovered in 2001 independently by our
group (187) and Hooper et al. (188). Even though both research groups utilized the
positional candidate cloning approach, following analysis were carried out differently.
Our group identified putative exons for KLK14, within the genomic area spanning the
KLK locus, using several gene prediction programs. Subsequent screening of the exons
against the human dbEST revealed one expressed sequence tag (EST) derived from lung
cDNA. The exon was experimentally confirmed by PCR, using various forward primers
located upstream of the putative exons and cDNA from several sources (187).
Alternatively, Hopper et al. screened the 300kb of draft sequence using the tBLASTN
algorithm with conserved peptide motifs spanning the catalytic triad of histidine, aspartic
acid, and serine residues of S1 family of serine proteases (WVLTAAHC, HDLMLLKL,
and GDSGGPL) (188). The acquired partial peptide sequence was used to search the
human dbEST. Two EST clones, derived from kidney and squamous cell carcinoma,
were obtained and was aligned against the sequence covering the 19q 13.4 region of
human genome. The 3′ UTR of the gene was further identified, using prostate cDNA
library (188).
Both research groups were able to identify identical coding region for KLK14,
however each group reported a different 5′ and 3′ UTR exons. According to the data of
Yousef et al. (187), KLK14 contains two non-coding exons within its 5′ UTR. This is
consistent to previous reports of 5′ UTR regions in other members of the family (187).
INTRODUCTION 38
However Hooper et al. (188) were unable to identify any UTR region in the 5′-end of
KLK14. Instead they identified a non-coding exon with the 3′ UTR.
1.3.2. Organization of KLK14 Gene and Protein Structure
KLK14 gene is located between KLK13 and Siglec-9 on chromosome 19q13.3-
13.4 and is transcribed from telomere to centromere. Similar to the remaining KLKs,
KLK14 codes for a putative serine protease with conserved catalytic triad codons and contains 5 coding exons and 4 intervening introns (Fig. 1.5)(187;188).
KLK14 is transcribed to ~1460bp mature mRNA, consisting of 267 bp of 5′ UTR,
756 bp open reading frame, and 436 bp of 3′ UTR. So far, two mRNA variant of KLK14 have been identified (187;188), however the functional relevance of these variants remains unclear.
The protein product of KLK14 contains 18 amino acid signal sequence that direct the newly synthesized protein to the endoplasmic reticulum for secretion, a 6 amino acid pro- sequence that is cleaved upon activation, and a 227 amino acid mature protein (189).
Proteolytic removal of the pro-sequence at Lys (24)- Ile(25) is required for activation of
KLK14 (189). As with all serine proteases within the S1A family, active KLK14 contains the catalytic triad of His (57)- Asp (102)- Ser (195). It also contains 12 conserved cysteine residues, forming 6 disulfide bonds, and a SYG motif that is believed to be critical in the correct orientation of the scissile bond of the substrate (Fig. 1.5) (189).
KLK14 has relatively high sequence similarity with other KLKs, particularly with
KLK6, 7, 8, 11, with ~ 47% identity (187). Phylogenetically, KLK14 has been clustered with KLK4 and 5, suggesting their common ancestry (33).
INTRODUCTION 39
1.3.3. Substrate Specificity
Given the structural features of KLK14, including the presence of Asp (198) in its
S1 binding pocket, KLK14 was initially predicted to possess trypsin-like substrate specificity with a preference for basic scissile bond at position P1 (Schechter and Berger
(190) nomenclature) (191;192). However, as mentioned previously, subsequent phage- display and chromogenic substrate studies, indicated a dual trypsin-like and chymotrypisn-like substrate specificity towards both basic, i.e. arginine and lysine, as well as hydrophobic, i.e. tyrosine, at P1 position (64;193).
So far a number of putative biological substrates of KLK14 have been inferred, suggesting a possible role of this enzyme in skin desquamation and cancer progression
(194). For instance, based on identified phage- displayed substrate motifs, several ECM molecules such as laminin, collagen type IV, and matrilin-4 have been postulated as putative KLK14 substrates (193). These substrates were further shown to be processed by
KLK14 in-vitro, suggesting a role in ECM digestion (189). In addition, as mentioned earlier, KLK14 has been implicated in skin desquamation through degradation of intercellular (corneo) desmosomal adhesion molecules (67).
A). INTRODUCTION 40
B).
C).
FIGURE 1.5. Gene and protein characteristics of KLK14. A). KLK14 gene contains 5 coding exons and 4 intervening introns with a conserved intron phase pattern of I, II, 0. B). KLK14 is synthesized as preproenzymes. C). The theoretical tertiary structure of mature KLK14, predicted by homology modeling. The ribbon plot is shown looking into the active site. The position of the autolysis loop is indicated.
INTRODUCTION 41
1.3.4. Expression Pattern and Cellular Localization
KLK14 is reportedly expressed primarily in the breast, prostate, skin
(90;187;188;195) both at the mRNA and protein level. Even though KLK14 mRNA was reported to be expressed in several different regions of central nervous system (e.g. brain, cerebellum, and spinal cord) (187), it was only detected in the midbrain at low level and was absent in other regions (90). The observed discrepancy could be due to mRNA instability or rapid degradation of the protein. Varying level of KLK14 was also detected in several bodily fluids, including seminal plasma, follicular fluid, sweat, vaginal fluid, serum, and amnionic fluid (90;189;196;197). According to several in-situ hybridization and immunohistochemical studies, KLK14/KLK14 localizes to the glandular epithelia of the prostate, ductal columnar epithelial cells of the mammary gland, and the eccrine sweat glands, stratum granulosum and stratum corneum of the skin (65;90;187;188;198).
1.3.5. Regulatory Mechanism of Proteolytic Activity
KLK14 activity is tightly regulated through various regulatory mechanisms, including internal cleavages, endogenous inhibitors, and ions (189). Analogous to several other members of the family, autodegradation and subsequent inactivation has also been proposed (189). Autodegradation was shown to begin with the proteolysis of the most solvent- accessible P1-arginine residues, leading to destabilization of the tertiary of the protein. Complete degradation of the enzyme is often followed. Furthermore, as mentioned previously, KLK14 is inhibited by several serpins such as PAI-1, AT, AP,
ATIII, and ACT (189). Lastly, KLK14 is believed to be regulated by citrate and zinc(189). Citrate was shown to enhance KLK14 activity, possibly through inducing a more active conformation in KLK14 (189). Conversely, even though no Zn2+-binding site
INTRODUCTION 42 has thus far been identified, zinc ion has been shown to inhibit KLK14 activity (189).
Given the high abundance of these ions in seminal plasma, ion-mediated inhibition of
KLK14 has been suggested to have physiological relevance (189).
INTRODUCTION 43
1.4. MALE REPRODUCTIVE SYSTEM
1.4.1. Semen Composition
Semen is primarily consisted of secretions from the accessory glands of the male
genital tract (199). Seminal vesicles and prostate gland accumulatively contribute to
approximately 90% of the total ejaculate, while the remaining 5% is formed by the
bulbourethral and urethral glands (199). Seminal vesicle secretion is the main constituent
of seminal plasma, affecting the function of sperms and the physiology of seminal
plasma. For instance, the fructose present in seminal vesicle secretion is an important
energy source of the spermatozoa. Sperm mobility is completely abrogated in the absent
of vesicular secretion due to insufficient nutrients and lower pH.
Prostatic gland is the second contributor of semen, accounting for 13-33% of total
volume of seminal plasma (199). Prostatic fluid is secreted directly into the urethra
through multiple ducts surrounding the verumontanum in the prostatic urethra (199).
Prostate secretion consists of mainly enzymes involved in semen clotting and
liquefaction, including vesiculase, proteases, peptidases, and hyaluronidase (199).
Prostatic secretions also contain ions, such as citrate, zinc, and magnesium. Citrate is
used as an indicator of prostatic function. The exact function of zinc and magnesium is
not fully understood. As mentioned previously, zinc is believed to play a role in
inhibiting proteolytic activity of certain seminal proteases, including KLKs.
INTRODUCTION 44
1.4.2. Semen Physiology
1.4.2.1. Sperm production and maturation
Spermatogenesis, the process of differentiation of testicular stem cells (TSCs) into mature spermatozoa, is initiated in the seminiferous tubules of the testes, which produce immature sperm cells (200-202). Subsequent maturation occurs during epididymal transition, where immature spermatozoa acquire motility and fertilizing capacity
(203;204). Sperm motility is characterized by a rhythmic, asymmetric three-dimensional movement of the flagellum (205). Even though sperm oscillations can originate from different regions of the flagellum, the basal region of sperm is postulated to act as a pacemaker, controlling the frequency of each beat (206). After ejaculation, sperms acquire a so-called “forward motility”, characterized by progressive, more forceful movement (205). Following entry to the female genital tract, through the process of capacitation, hyperactive sperms are developed (205). During this phase sperm gains a more energetic and less symmetric flagellar beat that could help sperm to progress through the cervical mucus, the oviduct, and the cumulus oophorus and finally penetrate to the zona pellucida of the oocyte (200;201;207).
To date, several extra- and intracellular factors have been implicated in the development and maintenance of sperm motility. Majority of regulatory components of sperm motility seem to exert their function through regulating various physiological aspect of sperm, including tyrosine phosphorylation of certain sperm- tail proteins, osmolarity and sperm volume (205). However, even though less emphasized, sperm motility could be affected indirectly, mainly through the physical constraint imposed by hyperviscous or sub-liquefied semen (208-210).
INTRODUCTION 45
1.4.2.2. Seminal clotting and liquefaction
Normally, human semen coagulates spontaneously upon mixing of its various glandular fractions in order to form a depository of spermatozoa in the rear vaginal cavity
(211-213). Subsequent liquefaction of coagulum within minutes (~5 to 20 minutes after ejaculation) allows for a progressive release of motile spermatozoa (211;214). Impaired liquefaction of semen has been reported to inversely affect sperm motility, as it creates physical hindrance (208-210).
Ejaculate constituents enter the posterior urethra in a specific order. The first fraction of the ejaculate consists of mainly spermatozoa accompanied by epididymal fluid
(199). Immediately after, the secretions of the prostate and then the secretions of the seminal vesicles are followed. Thus various components of the whole ejaculate only come into contact with each other only after they are propelled down the penile urethra during a process known as emission (199). However, only after liquefaction the complete mixing of the ejaculate can occur (199).
Liquefaction is achieved through a stepwise proteolytic cleavage of the gel proteins Sg I and II into soluble proteins, followed by their peptidic fragmentation. These peptides are eventually degraded into their constituent amino acid residues (215-219).
Semen coagulation/ liquefaction is under a tight regulatory control. For instance,
Sg proteins chelate with the excess of free Zn2+ immediately after ejaculation and
undergo structural modifications, inducing aggregate complex formation (220-224). Sg
degradation is mainly modulated through activation of KLK3 (159;223;225). As
mentioned previously, the enzymatic activity of KLK3 is tightly controlled through a
number of endogenous inhibitors and regulatory feedback loops. For instance, along with
INTRODUCTION 46
Sg proteins, the serine protease inhibitor PCI is secreted from the lumen of seminal vesicles (226). Recent evidence indicates that PCI complexes with Sg, preventing its pre- mature hydrolysis by active KLK3 (160). KLK3 activity is believed to be further inhibited by free Zn 2+ in prostatic secretions (159;161). Sg- chelation with free Zn 2+ results in an immediate drop in the available Zn 2+, which consequently leads to KLK3
activation. Conversely, Zn2+ is released gradually as Sg proteins are fragmented by
KLK3. The increased level of Zn 2+ serves as a negative feedback loop to prevent
excessive proteolysis that may damage the integrity of spermatozoa (227).
Recent evidence indicates an additional level of complexity in the regulation of
the proteolytic cleavage of Sg proteins. For instance, in-vitro data suggest that other proteases, particularly other members of the KLK family (i.e. KLK2 and KLK5), are directly or indirectly involved in Sg processing (154;194;228). In addition, emerging reports suggest a cascade-mediated protease activation mechanism, regulated by a number of positive and negative feedback loops. For example, as mentioned previously,
KLK5 is suggested to autoactivate and, in turn, activate proKLK3 (51). Likewise, even though still controversial, KLK2 has been suggested to activate proKLK3 (155-157).
1.4.3. Sperm Transport in the Female Reproductive Tract
Seminal plasma was long seen merely as a medium required for survival and transport of sperms. Emerging evidence however points to an additional role of various
components of the seminal plasma in regulating molecular and cellular changes in the female body to facilitate conception and pregnancy (229).
INTRODUCTION 47
1.4.3.1. Postmating inflammatory responses
Immediately after insemination, seminal plasma induces a rapid and transient influx of inflammatory cells to the site of semen deposition (229). These post-mating inflammatory responses are mainly mediated through synthesis of pro-inflammatory cytokines, including GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-
(interleukin-) 6, and various chemokines (229). These pro-inflammatory factors induce an array of cellular changes leading to the extravasations of infiltrating macrophages, dendritic cells, and granulocytes into the subepithelial stromal tissues of the female reproductive tract (229).
Seminal plasma was postulated to regulate several reproductive processes through recruiting various leukocytes during the pro-inflammatory response phase. These leukocytes are involved in the four main processes of (1). clearance of defective sperms and microorganisms introduced into the uterus (2). tissue remodeling to increase endometrial receptivity (3). activation of cytokines and growth factors required for embryo development, prior to the implantation (4). activation of immune responses specific to semen antigens and other parental transplantation proteins (229).
1.4.3.2. Role of semen in maternal immune tolerance
Sperm cells are extremely unique in that they must physiologically survive transplantation into a foreign host in order to successfully penetrate and reach the oocytes. Given the highly potent immune system present in the female genital tract, seminal plasma has long been proposed to employ various immunosuppressive strategies to attenuate the immune attack induced by sperm entry. For instance to overcome the
INTRODUCTION 48 complement system of innate immunity, a number of complement regulators have been proposed in both seminal plasma and on the spermatozoa (230). Clusterin is the most abundant complement inhibitor identified thus far (231). Clusterin is produced in the seminiferous tubules and seminal vesicles (232). Interestingly, the level of clusterin in seminal plasma is clinically proportional with the fertilization capacity of the semen in in-vitro fertilization (233). Analogous to its function in blood plasma, clusterin is also an inhibitor of the membrane attack complex (MAC) of the complement system (233). Other complement inhibitors identified more recently include the CReg CD 46, CD55, and CD
59 (234-236).
1.4.3.3. Immune regulatory function of TGFβ in seminal plasma
More recently, TGFβ has been implicated as a key immune regulatory protein in
human seminal plasma (237). Seminal plasma contains a large amount of TGFβ,
approximately five-folds more than serum TGFβ (237). Majority of human seminal
TGFβ is synthesized in male accessory glands, particularly in the prostate (238).
The immune regulatory function of TGFβ is complex and paradoxical, depending
on the microenvironment and the nature of target cells. The dual nature of TGFβ is most
evident in inflammatory responses induced in the female reproductive system, where it
initially acts as a pro inflammatory agent. This effect is very transient and is replaced by
the ability of this cytokine to skew immune response to increase the receptivity of the
female tract to allogentic sperms. The immune suppressive function of TGFβ is mainly
mediated by regulating the maturation and proliferation of type2 or Th3 T-lymphocytes
(237).
INTRODUCTION 49
Seminal TGFβ was first identified as a key immunosuppressive cytokine.
Fractionation of human seminal plasma by gel filtration revealed a fractions of 100 to greater than 440kDa with strong immunosuppressive properties, as determined by their ability to kill the activity of interleukin-2 stimulated lymphocytes (239). Upon further characterization of the fractions, TGFβ was identified as the primary immunosuppressive protein (239). Thus far, five isomers of the family have been identified, with three of them expressing in mammalian cells (240). Human seminal plasma contains TGFβ1 and
TGFβ2, mainly in their latent form (241). TGFβ1 is expressed in approximately 10 folds higher, averaging 240 ng/ml (241). More than 50% of the total immune tolerance property of seminal plasma has been attributed to the biological activity of TGFβ1 (237).
Biologically active TGFβ family contains homodimeric proteins with molecular weight of approximately 25 kDa (241). The dimmers are linked with disulfide bonds and are often associated noncovalently with high molecular weight proteins, in their latent
(inactive) form (241). TGFβ proteins undergo several posttranslational “ processing” steps prior to secretion, including glycosylation, mannose-6-phosphorylation of the glycoprotein and disulfide isomerization (242-244). In addition, during transition through the rough endoplasmic reticulum, a signal sequence of 29 amino acids is cleaved (242).
Activation is achieved by cleavage of the N-terminal latency- associated peptide (LAP) at
Arg(278), followed by its further cleavages to expose the receptor binding motif of mature TGFβ (245;246). LAP contains three N-linked glycosylation sites, which may be instrumental in maintaining the latent state, as endoglycosidase F treatment is reportedly lead to activation of TGFβ (247). In addition, the disulfide-bond linking the dimeric LAP is essential for latency, since mutation in cysteine residues at position 223 and 225 almost
INTRODUCTION 50 completely abolished activity (248). Latent TGFβ contains an additional component, known as latent TGFβ binding protein (LTBP) (249). Members of the LTBP family, in particular LTBP1, has been implicated in several aspects of regulating latent TGFβ by facilitating its secretion, modulating its activation, or releasing it from matrix- bound storages (250).
Even though several harsh environmental conditions, including acidic (pH of less than 3.5), heat, and low dose gamma irradiation has been shown to release TGFβ from latent TGFβ (251;252), physiologically relevant activator components are largely unknown. A number of processes, including deglycosylation, proteolysis by calpain, cathepsin, thrombospondin- 1, KLK3 (PSA), and plasmin, as well as exposure to reactive oxygen species have been suggested as potential physiologic activators of TGFβ (253). It is still unclear if any of these mechanisms are utilized in physiological conditions in-vivo.
Interestingly, TGFβ present in mouse uterine fluids after insemination was found to exhibit more than 70% biological activity, while only less than 30% of TGFβ recovered from seminal vesicle was active (237). This suggest that activation of seminal
TGFβ occurs physiologically only after ejaculation or after deposition in the female tract.
So far, a number of serine proteases, including plasmin, substilisin-like endoproteases, tissue plasminogen activator, and urokinase plasminogen activator, were reported to proteolytically activate seminal TGFβ1 (254).
INTRODUCTION 51
1.4.4. Male Factor Infertility
Approximately 15-20% of couples within reproductive age are expected to experience difficulties in achieving pregnancy (255). It is estimated that an isolated male factor is associated with roughly 30% of these cases, while a combination of male and female factors contribute to an additional 20% (255). Thus, abnormal male factors are estimated to be involved in approximately half of cases in which couples seeking infertility treatment. Despite the significant role of male factors in couples’ infertility, research in this area has lost much of its impetus, particularly since the advent of intracytoplasmic sperm injection (ICSI) in early 1990 (256;257). In recent years, however, it has become evident that a shift of focus is crucial towards achieving natural pregnancy through accurate diagnosis of male infertility, in order to reduce the cost and emotional predicament of assisted conception.
1.4.4.1. Male infertility diagnosis
Given that male fertility is essentially dependent on fertilization potential of sperms, semen analysis to assess sperm quantity and quality is instrumental in the diagnosis and treatment of male-related sub- or infertility. The idea of assessing male fertility potential through a basic semen analysis was first proposed in 1677 by van
Leeuwenhoek, following the Johan Ham’s discovery of human spermatozoon (258).
However, the first systematic approach was not developed until the end of the 19th
century, when the fist report of sperm counting using hemocytometer was published by
Lode in 1891 (258). Subsequently, Hotchkiss developed the first grading system of sperm
motility in 1941, which was later modified by MacLeod and Heim to incorporate
INTRODUCTION 52 motility and progressive activity parameters (258). The following research of Belding,
Williams et al., McLeod, Gold and Freund, and Eliasson on sperm morphology, the current specification of “normal” semen variables was nearly completed. In order to obtain a uniform result from semen analysis, the first WHO (world health organization) manual on standardized semen variables was published in 1980 (259). Guidelines to the diagnosis and management of male factor infertility are routinely updated by the WHO as new findings become available.
The first step in performing semen analysis involves the examination of factors describing the overall appearance of semen, including the color and odor, coagulation and liquefaction, viscosity, and pH (260). Coagulation and liquefaction are two very essential aspects of semen analysis that is often overlooked by investigators, mainly because of the inconvenience of semen production at the site of examination. Lack of coagulation may be an indication of congenital absence of the vas deferens and seminal vesicles. For this reason, incomplete coagulation is often accompanied by a reduced level of fructose in the seminal plasma (261). Similarly, incomplete or lack of liquefaction could severely affect sperm motility as it prevents the release of motile spermatozoa from the coagulum (258).
Aberrant liquefaction is often a sign of prostate dysfunction, usually as a result of previous prostatitis (258). Increased viscosity may be due to various factors, including abnormal prostate function due to an infection in the genital tract, prostate, or seminal vesicles (261;262). Hypervisous semen is considered as one of the causes of subfertility for in-vivo conception and can also interfere with accurate determination of spermatozoa concentration and motility (262).
INTRODUCTION 53
The basic physical examination of semen is often followed by a more detailed microscopic analysis to quantitate various semen parameters, including motility and forward progression, sperm concentration, and to evaluate sperm morphology (260).
Sperm motility is often measured using automated computer-assisted semen analysis
(CASA) (258). Sperms are classified based on their forward progressive motility into four grades a-d, with grade “a” having rapid progressive motility and grade “d” being immotile (258). This definition however varies, depending on whether the evaluation is done at room temperature or at 37°C. For the latter, a minimum of 25 µm/sec is required for the grade a category, while the cutoff at the room temperature is 20 µm/sec (258).
INTRODUCTION 54
1.5. AIM OF THE PRESENT STUDY
1.5.1. Rationale
Proteolytic cascades function to transduce signals through sequential activation of
protease zymogens, enabling cells to respond to environmental cues (5). The key
characteristic of a proteolytic cascade is the rapid and highly controlled amplification of
active executor proteases in response to minute amount of initiator enzymes. This way,
deleterious damages due to prolonged or excessive proteolytic activity is prevented.
All KLKs, except KLK4, require cleavage after arginine or lysine for their
activation. Given that these are preferred trypsin-like cleavage sites, KLK activation is
mediated only by trypsin-like enzymes. This suggest a linear pattern of activation at least
in case of chymotrypisn-like KLKs (194). In addition, KLKs tend to co-express in
varying levels and coordinately dysregulated in various pathologic conditions, suggesting
common regulatory mechanisms.
To date, several in-vitro proteolytic cascades have been suggested within a few
members of the KLK family. Yet, the physiologic relevance of these cascades remains to
be fully elucidated. Given the co-expression of these KLKs in seminal plasma or skin,
these cascades have been implicated in skin desquamation and/or semen liquefaction.
However, several other KLKs have been reported in the skin and seminal plasma
(65;66;162) and may participate in common proteolytic cascades.
Recent evidence suggests that KLK14, a newly characterized trypsin-like KLK, is
possibly a key protease in the skin, contributing to approximately half of the total trypsin-
like proteolytic activity in the SC layer. In addition, Zn2+ has been shown to strongly
INTRODUCTION 55 inhibit KLK14 enzymatic activity, suggesting a potential role of the protein in seminal plasma.
1.5.2. Hypothesis
Given that KLK14 a). is a novel trypsin-like protease, b). exhibits high enzymatic activity both in its recombinant and endogenous form, and c). is expressed primarily in seminal plasma and the skin, we hypothesize that KLK14 mediates various patho(physiological) processes at the main sites of its expression through highly orchestrated proteolytic cascades.
1.5.3. Objectives
1. To identify putative KLK14- mediated cascade(s) through screening of a
heptapeptide library of KLK activation motifs.
2. To validate the theoretical cascade model(s) in relevant biological system(s).
3. To define the clinical utility of the members of the cascade(s) in identified
biological system(s).
4. To investigate potential role(s) of the cascade(s) in novel physiological
processes.
PUTATIVE KLK14-MEDIATED CASCADES 56
CHAPTER 2 Identification of Potential KLK14- Mediated Cascade(s)
Sections of this chapter were published in the Journal of Biological Chemistry:
Emami N and Diamandis EP. Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin. J Biol Chem. 2008; 283(6):3031-41.
Copyright permission has been granted.
PUTATIVE KLK14-MEDIATED CASCADES 57
2.1. INTRODUCTION
Emerging evidence indicates that KLKs are activated in a step-wise manner,
which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. In addition, many members of the KLK family have been reported to be active in seminal plasma and/or skin,
suggesting their involvement in common proteolytic cascades. KLK14 in particular, is
highly active and has recently been proposed as one of the key trypsin-like proteases
involved in skin desquamation.
In an attempt to delineate the possible involvement of KLK14 in KLK activation
cascades, this study examines the interaction between this enzyme and other members of
the family, using an unbiased library of activation motifs of the fifteen KLKs and further
verifies those that are known to be expressed in skin and/or seminal plasma.
PUTATIVE KLK14-MEDIATED CASCADES 58
2.2. EXPERIMENTAL PROCEDURES
2.2.1. Materials
The synthetic heptapeptides N-Ile-Gln-Ser-Arg-Ile-Val-Gly-C, N-Ile-Leu-Ser-
Arg-Ile-Val-Gly-C, N-Ser-Cys-Ser-Gln-Ile-Ile-Asn-C, N-Ser-Ser-Ser-Arg-Ile-Ile-Asn-C,
N-Glu-Gln-Asn-Lys-Leu-Val-His-C, N-Gln-Gly-Asp-Lys-Ile-Ile-Asp-C, N-Gln-Glu-
Asp-Lys-Val-Leu-Gly-C, N-Asp-Thr-Arg-Ala-Ile-Gly-C, N-Asn-Asp-Thr-Arg-Leu-Asp-
Pro-C, N-Glu-Thr-Arg-Ile-Ile-Lys-C, N-Ala-Thr-Pro-Lys-Ile-Phe-Asn-C, N-Glu-Ser-Ser-
Lys-Val-Leu-Asn-C, N-Asp-Glu-Asn-Lys-Ile-Ile-Gly-C, N-Asp-Gly-Asp-Lys-Leu-Leu-
Glu-C were purchased from Genemed Synthesis (San Francisco, CA, USA) and were diluted in water and stored at -20 °C. The synthetic substrates, Suc-Arg-Pro-Tyr- pNA.HCl (RPY-pNA), Pro-Phe-Arg-AMC (PFR-AMC) and D-Val-Leu-Lys-Thiobenzyl
ester (VLK-SBzl), were purchased from BACHEM (King of Prussia, PA), Pharmacia
Hepar-Chromogenix (Franklin, OH, USA), and Chromogenix (Milano, Italy),
respectively. Recombinant proKLK3 produced in E.Coli, was a gift from Spectral
Diagnostic Inc (Toronto, ON, Canada). Mature KLK1, produced in a baculovirus/insect cell line system, was kindly provided by Dr. M. Blaber (Florida State University, USA).
KLK14 and KLK11 were produced in house, as described previously (263). HUK-IgG, an antibody recognizing KLK1, was kindly provided by Prof. J. Chao (Medical
University of South Carolina, USA).
2.2.2. Heptapeptide Library Screening
25 µg of heptapeptides were incubated at 37oC with 1 µg of KLK14 at 1,500:1
molar ratio in KLK14 assay buffer (100mM phosphate buffer, 0.01% Tween 20, pH 8.0),
in total volume of 200 µl. Reactions were stopped at different time points by freezing the
PUTATIVE KLK14-MEDIATED CASCADES 59 samples with liquid nitrogen. A 150 µl aliquot of each time point was diluted 2- fold with loading buffer (0.1%TFA in H2O). A scrambled heptapeptide (Hep0; of random
sequence) was included as a negative internal control to account for experimental
variations.
Probable hits, i.e. heptapeptides cleaved by KLK14, were identified using reverse
phase- high performance liquid chromatography (RP-HPLC). LC separation was carried
out using an analytical C18 column (TOSOH) and a mobile phase consisting of 0.1%TFA
in H2O (Buffer A) and 0.1%TFA in ACN (Buffer B). Samples were eluted with a linear gradient of 0 to 60% of buffer B at a flow rate of 0.8ml/min. Retention times of heptapeptides were measured prior to incubation with KLK14. Absorption (214nm) of peaks representing the remaining uncleaved heptapeptides were recorded at different
incubation time points and normalized to the corresponding value of Hep0. Cleavage
efficiency was calculated as a percent height (mAU) reduction in the absorption of the
remaining uncleaved fragments.
Positive hits, i.e. heptapeptides with cleavage efficiency of 85% or higher (within
five hours) were selected for further verification. Cleavage sites were verified by tandem
mass spectrometry. Sample separation was replicated as explained above and scanned,
using an API 3000 triple quadrupole mass spectrometer (MDS Sciex). The HPLC was
conducted using an Xterra C18 column (3.0X50 mm, 2.5 µm) with mobile phase
consisting of 50% ACN containing 0.5% TFA in isocratic mode. The m/z ratios
corresponding to the doubly and/or singly charged daughter fragments were extracted
from the total ion current (TIC) scans. Collision energy (CE) of 17 volts was applied to
PUTATIVE KLK14-MEDIATED CASCADES 60 further break extracted peptides. Peptide sequences were extrapolated from extracted ion chromatograms (XICs).
2.2.3. Recombinant KLK1 Production
The full-length coding region of KLK1 protein [Genebank accession no.
AAH05313] was PCR- amplified and cloned into the pcDNA3.1(-) (Invitrogen) mammalian expression vector at EcoRI and XbaI sites. Recombinant clones were stably transfected in the human embryonic kidney cell line, HEK293. Positive clones were selected by their ability to survive serial passages in Geneticin. The clone expressing the highest amount of KLK1 was selected. Seeding density, cell number, and harvest time were optimized to maximize protein production with minimal cell death .
o The recombinant clone was grown in a humidified incubator at 37 C and 5% CO2
in MDEM culture medium (Gibco) supplemented with 10% FBS. Approximately
180x106 cells were seeded into ten 175cm2 tissue culture flasks and grown to 60-70%
confluency. The media was replaced with CDCHO serum-free media (Gibco),
supplemented with 8mM glutamine, and incubated for 7 additional days. Cell supernatant
was collected and frozen at -80oC until further use.
Purification was achieved, using anion-exchange fast protein liquid
chromatography (FPLC). Cell supernatant was concentrated 10 times and loaded onto a
Hi-Trap DEAE-FF anion exchange column (Amersham Biosciences). The column was
eluted with a linear gradient of 0%-80% of 20mM Tris +1M NaCl pH 8.0 (Buffer A) at a
flow rate of 3 ml/min. Fractions were analyzed by an in-house enzyme-linked
immunosorbent assay (ELISA) and those containing KLK1 were pooled. Further
PUTATIVE KLK14-MEDIATED CASCADES 61 purification using RP-HPLC was carried out using C8 reverse phase column, with a step gradient of 0-100% of 0.1%TFA in ACN, described above.
The enzymatic activity of recombinant KLK1 was tested using the fluorogenic substrate PFR-AMC.
2.2.4. Activation of ProKLK3 and ProKLK11 by KLK14
Activation was monitored as an increase in the absorbance of RPY-pNA in KLK3 optimized assay buffer (0.1mM Tris, 3mM NaCl, 0.01% Tween 20, pH 7.5) and VLK-
SBzl in KLK11 optimized assay buffer (50mM Tris, 1M NaCl, 10mM EDTA, pH 8.5, containing 0.1mM DTNB), in total volume of 200 µl. ProKLK3 and proKLK11 were added to active KLK14 at various molar ratios and incubation times at 37oC in KLK14
optimized activity assay buffer (100mM phosphate buffer, 0.01% Tween 20, pH 8.0),
total volume of 50 µl . Digestions were repeated three times.
Absorbance was measured on a Wallac Victor Fluorometer (PerkinElmer Life
Sciences) at 405nm for KLK3 and 420nm for KLK11. In case of KLK3, the background
absorbance and residual activity of KLK14 was subtracted from raw values of enzyme
alone and reaction mixtures, respectively. The residual activity of proKLK3 was
accounted for by including an additional proKLK3 alone reaction. In contrast, given the residual activity level of KLK11 and the very low KLK14 activity towards VLK-SBzl, the background absorbance and residual activity of KLK11 was subtracted from raw values of enzyme alone and reaction mixtures, respectively.
KLK14-mediated fragmentation of proKLK3 was determined by incubating proKLK3 with active KLK14 at 10:1 molar ratio for varying time points, in total volume of 50 µl. Identical reactions were run in two separate SDS-PAGE gels (1:4 ratio) under
PUTATIVE KLK14-MEDIATED CASCADES 62 reducing conditions. One gel was silver-stained and the gel with 4 times more sample was electroblotted to polyvinylidene difluoride (PVD) membrane and stained with
Coomassie Blue stain. Fragments were cut from the membrane and N-terminally sequenced.
2.2.5. Activation of ProKLK1 by KLK14
ProKLK1 was added to active KLK14 at various time points at 1:1 molar ratio, in total volume of 50 µl, at 37oC. Reactions were repeated in duplicates. KLK1-specific
activity was measured by fluorescence release of the pulled down KLK1 protein. 200 ng of KLK1- specific polyclonal antibody (HUK-IgG) were immobilized overnight on a 96- well polystyrene plate in coating buffer (50mmol/L Tris, 0.05% Tween 20, pH 7.8). The plate was washed two times with washing buffer (50mmol/L Tris, 150 mmol/L NaCl,
0.05% Tween 20; pH 7.8).
Reaction mixtures were loaded into each well, incubated for 2 hours with shaking, and washed six times with the washing buffer (above). Subsequently, 0.25mM of PFR-
AMC in KLK1 optimized activity assay buffer (20mM Tris/HCl, pH 9.0, 1mM EDTA,
10% DMSO, and 0.1% TritonX-100) was added to each well. Increase in fluorescence signal was measured on a Wallac Victor fluorometer, set at 355nm for excitation and
460nm for emission. Basal activity of both KLK1 and KLK14 were measured at time zero and subtracted from raw values. Reaction rates (FU/min) correspond to the slope of the fluorescence release-time plot.
PUTATIVE KLK14-MEDIATED CASCADES 63
2.2.6. N-terminal Sequencing
Sequencing was performed with the Edman degradation method.
PUTATIVE KLK14-MEDIATED CASCADES 64
2.3. RESULTS
2.3.1. Heptapeptide Screening
In an attempt to identify potential downstream targets of KLK14, a library of 15
heptapeptides representing the putative P4-P3-P2-P1-P'1- P'2-P'3 positions of active
motifs of KLKs was designed (Table 2.1). Heptapeptides were incubated with the
recombinant active KLK14 for various time intervals. Cleavage was monitored by RP-
HPLC. Cleavage efficiency was calculated as a percent mAU reduction in the peak
representing the undigested peptide, normalized to that of Hep0 (Fig. 2.1). Cleavage
specificity was determined by LC-MSMS of the two daughter peaks, representing the P4-
P1 and P'1- P'3 fragments (Fig. 2.2).
KLK14 cleaves heptapeptides representing KLK1, KLK2, and KLK3 pro-
peptides with high efficiency. Heptapeptides for KLK 5, 7, 11, and 12 were digested with
moderate (≥ 85% digest after 5hrs) to low efficiency (≤ 85% digest after 5hrs), while
heptapeptides for KLK 4, 6, 8, 9, 10, 13, 14, and 15 were not cleaved at all (Table 2.1).
Given the rapid nature of proteolytic cascades, we only considered screening hits
with high to moderate cleavage efficiency. These results are consistent with the
previously reported KLK14- mediated activation of KLK5 (64).
2.3.2. Activation/Deactivation of ProKLK3 and ProKLK11
Given the high cleavage efficiency of heptapeptides representing the pro-peptides
of proKLK3 and proKLK11, we examined whether these proteins function as immediate
downstream targets of KLK14. The ability of KLK14 to activate recombinant, pro-forms
of these proteins was tested.
PUTATIVE KLK14-MEDIATED CASCADES 65
Table 2.1. Relative cleavage efficiency of heptapeptides by active KLK14
% Digestion Heptapeptide sequence1 ProKLK 1h 3h 5h
IQSR↓IVG KLK1 97 99 99
IQSR↓IVG KLK2 97 99 99
ILSR↓IVG KLK3 87 95 97
SCSQ↓IIN KLK4 0 0 0
SSSR↓IIN KLK5 57 82 88
EQNK↓LVH KLK6 0 0 0
QGDK↓IID KLK7 41 52 64
QEDK↓VLG KLK8 0 0 0
DTR↓AIG KLK9 0 0 0
NDTR↓LDP KLK10 0 0 0
ETR↓IIK KLK11 55 77 90
ATPK↓IFN KLK12 19 48 62
ESSK↓VLN KLK13 0 0 0
DENK↓ IIG KLK14 0 0 0
DGDK↓ LLE KLK15 0 0 0
1 Heptapeptides were designed to encompass the activation sites of each KLK (denotd by arrow). All sequences are shown in the N→ C direction and with single letter amino acid designations.
PUTATIVE KLK14-MEDIATED CASCADES 66 A).
B).
FIGURE 2.1. Monitoring of heptapeptide (Hep) cleavage. 25 µg of Hep1 were incubated with recombinant active KLK14 at 1500:1 molar ratio at A). 0h and B).1h. The cleavage was monitored by RP-HPLC . Hep1N and Hep1C represent the two daughter fragments generated after digestion the full-length Hep 1. The scrambled sequence Hep0 was used as an internal negative control. Cleavage sites were verified by mass spectrometry. Dashed lines show gradient profile as described in text.
PUTATIVE KLK14-MEDIATED CASCADES 67 A).
B).
C).
continued…
PUTATIVE KLK14-MEDIATED CASCADES 68
FIGURE 2.2. Validating the cleavage specificity. The cleavage site of Hep1digested with KLK14 was determined by LC-MSMS, using the API 3000 triple quadruple mass spectrometer. The mass spectrometer monitored the ion transitions of A). Hep1 at m/z 387.2→531.4, B). Hep1N, 252.6→391.2 and C). Hep1C, 288.7→175.1. The LC-MSMS method was developed using synthetic Hep 1, Hep 1N and Hep 1C. The collision energy (CE) was set at 17 v.
PUTATIVE KLK14-MEDIATED CASCADES 69
Since KLK3 exhibits specificity towards chymotrypsin-like substrates, the
chromogenic synthetic tripeptide RPY-pNA with chymotrypsin-like specificity was
employed. KLK3 activation was dependent on the enzyme to substrate molar ratio (Fig.
2.3A).
Characteristic to proteolytic cascades, the activation seemed to be rapid and
transient; KLK14 activated proKLK3 within the initial five minutes. KLK3 enzymatic activity was incrementally amplified over the next thirty minutes (Fig. 2.3B). However,
the reaction rate declined following longer incubation (Fig. 2.3C), suggesting a
deactivation mechanism that may act as a negative feedback loop regulating the
proteolytic activity of KLK3.
This observation was confirmed by sequencing the cleaved fragments (Fig. 2.3D).
Bands a, b, and c have the N-terminal sequence of IVGGWE (the sequence of active
KLK3), indicating KLK14-mediated activation of proKLK3. In addition, two bands with sequence SGWGS (band d), cleaved after tyrosine 130, and KLQCVD (band e), cleaved after lysine 145, were detected. These bands represent internal cleavages, leading to inactivation of activated KLK3.
PUTATIVE KLK14-MEDIATED CASCADES 70 A).
B). C).
D).
FIGURE 2.3. KLK14- mediated regulation of proKLK3 activity. A). Molar ratio- dependent activation. ProKLK3 was incubated with 0.085 µM of active KLK14 in varying molar ratios of 10, 20, 50, and 100 for 15 min, at 37oC. B, C). Time- dependent activation/ deactivation. 0.2 µM of proKLK3 was incubated with KLK14 at a molar ratio of 1:10 for varying time intervals, at 37oC. Activity was monitored through cleavage of 1mM of the RPY-pNA substrate. Note the gradual increase in the absorbance within the initial thirty minutes followed by reduction of activity up to 3 hours afterwards. KLK3 line represents negative control (no KLK14 added) D). proKLK3 fragmentation by KLK14. ProKLK3 (0.85 µM) was incubated with KLK14 (0.085 µM) for 1 hour and visualized by silver staining. The N-terminal sequence of the fragments was identified by Edman sequencing.
PUTATIVE KLK14-MEDIATED CASCADES 71
Similarly, KLK14- mediated activation of proKLK11 was determined using the
VLK- SBzl substrate. KLK14 alone exhibited a low preference for the above substrate
(with approximately 75% less absorbance after 20 minutes of substrate incubation) as compared to KLK11, which was activated for 15 minutes with KLK14. Even though
cloned in its pro-form, our recombinant KLK11 exhibited a low basal activity (data not
shown), which was subtracted from the absorbance readings of the reaction mixtures.
KLK11 was activated within 2 minutes of incubation with active KLK14, in a both dose- and time- dependent manner (Fig. 2.4A and 2.4B, respectively).
PUTATIVE KLK14-MEDIATED CASCADES 72
A).
B).
FIGURE 2.4. Activation of proKLK11 by KLK14. A). Time dependent activation. 0.2 µM of proKLK11 was incubated with 0.02 µM of KLK14 for varying time intervals, at 37oC. B). Molar-ratio dependent activation. 10nM of KLK14 was incubated with proKLK11 in varying molar ratios of 5, 10, and 20 for 5 min, at 37oC. Activity was monitored by cleavage of 1mM of the VLK-SBzl substrate. Note the increase in absorbance, indicative of increased enzymatic activity of KLK11. The line representing KLK14 was obtained without proKLK11 addition. The basal activity of KLK11 alone has been subtracted.
PUTATIVE KLK14-MEDIATED CASCADES 73
2.3.3. Cloning, Expression, and Purification of Recombinant ProKLK1
As suggested by the library screening (Table 2.1), proKLK1 is a candidate target, activated by KLK14. To confirm KLK14- mediated activation of proKLK1, KLK1 protein was produced recombinantly. Stable HEK293 cell lines, expressing proKLK1 were generated. One of the clones with the highest expression was chosen for further study. The highest expression was observed at day 10 of culture in serum-free media.
Samples were FPLC- fractionated. Fractions 14- 23 contained varying amounts of KLK1, as determined by a KLK1- specific ELISA and silver staining. Pooled fractions were further purified by RP-HPLC.
Unfortunately, pure recombinant proKLK1 could not be isolated, possibly due to autodegredation and/or internal cleavage of the protein in the supernatant (data not shown). The purified recombinant KLK1 was determined to be enzymatically active, with a reaction rate of approximately 2,000 FU/min, using 120nM of recombinant pulled- down KLK1 and 1mM PFR-AMC substrate (data not shown).
2.3.4. Activation of KLK1 by KLK14
One of the hurdles in kallikrein research has been the lack of specific activity assays, due to overlapping substrate specificities of the majority of KLKs. Here, we developed a “sandwich- type assay” (Fig 2.5A) to measure specific enzymatic activity of several trypsin-like KLKs, including KLK1, with a detection limit as low as 30nM (data not shown). In this assay, the desired KLK is pulled down in microtiter plates. The activity can then be measured using a non-specific substrate. Given the high protein similarity between KLKs, it is important to avoid non-specific pull down due to antibody cross-reactivity. Figure 2.5B shows the pull down specificity of the KLK1 antibody
PUTATIVE KLK14-MEDIATED CASCADES 74
HUK-IgG, using recombinant mature KLK1 and 14. While soluble (non-immobilized)
KLK14 exhibited even a higher reaction rate towards the PFR-AMC substrate (Fig 2.5C), almost no enzymatic activity was observed for the pulled down KLK14 on KLK1 antibody-coated plates, using the same amount of substrate. Using this assay, we confirmed that KLK1 was activated by KLK14 in a time-dependent manner (Fig. 2.5D).
Based on the information provided above, a cascade model for seminal plasma and skin (Fig. 2.6 and 2.7, respectively) was developed. For seminal plasma, the cascade is based on six KLKs which have already been found at appreciable amounts in this fluid.
For skin, the cascade is based on five KLKs, known to be expressed in this tissue. It is conceivable that other KLKs and/or other classes of enzymes, as well as additional inhibitors, may also participate in such pathways.
PUTATIVE KLK14-MEDIATED CASCADES 75
FIGURE 2.5. Activation of proKLK1 by KLK14. A). Schematic presentation of the activity assay. The activated KLK is pulled downed. Background due to non-specific binding is reduced through a series of stringent washes. The activity of the activated KLK is measured by monitoring the fluorescence release of a non-specific substrate and normalized to the background signal. B). Specificity of the KLK1-sandwich pull down assay. KLK14 reaction rate was almost zero, when pulled down with anti-KLK1 antibody, ensuring assay specificity. C). To ensure that the mature form of both KLKs had a comparable enzymatic activity prior to the pull down, reaction rates of soluble (non-coated) 12nM of mature KLK1 and KLK14 were measured D). Time- dependent activation. 0.2 µM of KLK1 were incubated with 0.2 µM of KLK14 for 0, 10, 30, and 60 min. at 37oC. KLK1 was pulled-down in 96 microtiter plates, as described in panel A. Activity of the pulled-down KLK1 was monitored by cleavage of 0.25mM of the PFR- AMC substrate. The basal activity of KLK1 alone was subtracted, prior to calculating reaction rates.
PUTATIVE KLK14-MEDIATED CASCADES 76
FIGURE 2.6. Schematic presentation of proposed kallikrein cascades in seminal plasma. KLK2 and 5 autoactivate and along with active KLK14, activate pro-KLK3. KLK5 could also activate proKLK2 and proKLK14, as shown previously. Activated KLK3 acts as an executor protease in the liquefaction of seminal clot and release of spermatozoa through proteolytic processing of Sgl I/Sgl II. The cascade is regulated by a 2+ number of endogenous inhibitors (PCI, ATIII, ACT, and α2-M), Zn , collectively shown as “Inh”, as well as by (auto)degredation of active KLKs. Active KLK14 can further activate proKLK11 and KLK1, functions of which remain to be further elucidated. Zn 2+ binds to active KLKs and inhibits their activity. SgI and II along with FN form the semen coagulum at the time of ejaculation, entrapping motile spermatozoa. Immediately after ejaculation, Sgs chelate Zn2+, rendering KLKs active. Active KLKs, in turn, engage in the above proteolytic cascades, rapidly amplifying active KLK executors. Sgs and FN are subsequently degraded by the executor KLKs, resulting in semen liquefaction and release of motile sperm cells. Question mark indicates unknown function.
PUTATIVE KLK14-MEDIATED CASCADES 77
FIGURE 2.7. Schematic presentation of proposed kallikrein cascades in skin. KLK5 autoactivates and activates KLK14 and 7. Activated KLK14 activates proKLK1 and 11. Cascades are postulated to be triggered by SC acidification of the superficial layer of skin. Executor KLKs function in skin desquamation through degredation of the corneodesmosomal proteins, i.e. DSG1, DSC1, and CDSN. Desquamation is regulated by various serine protease inhibitors, such as SLPI, elafin, and certain LEKTI domains, positive feedback loops, and internal cleavages. The question mark indicates unknown function. For more definitions, please refer to our non-standard abbreviations.
PUTATIVE KLK14-MEDIATED CASCADES 78
2.4. DISCUSSION
The idea of proteolytic cascades in KLKs came into prominence only recently,
with accumulating evidence indicating their step-wise activation mechanism. For
instance, with the exception of KLK4, KLKs are activated by cleavage after lysine or
arginine, which are preferred trypsin-like cleavage sites. However, some of the KLKs are
chymotrypsin-like and thus require other trypsin-like proteases for their activation. As
mentioned previously, the chymotrypsin-like enzymes KLK3 and KLK7 were shown
experimentally to be activated by the trypsin-like KLK5.
Moreover, in tissues, KLKs are often expressed in groups at varying levels (162).
Assuming that enzymatic activity is proportional to the expression level of each KLK,
such co-expression patterns may further indicate hierarchical activation networks,
consisting of initiators, progressors, and executors. For example, KLK14 is expressed at
an average concentration of 5 µg/L in seminal plasma, while certain other seminal KLKs,
including KLK1 and 11, have a 10-103- fold higher expression levels (162). Moreover,
consistent with the proposed hierarchical model of cascades, seminal KLK3 is expressed
at the staggering rate of grams per litter (162) and functions as the key executor of Sg
hydrolysis during clot liquefaction (159;218;264). Similarly, KLK11 expression in skin is approximately 9 times higher, compared to KLK14 (162), further suggesting the notion
of activation networks and sequential zymogen activation.
In the case of seminal plasma, additional evidence reinforcing the idea of
proteolytic cascades comes from the striking overlap between regulatory components of
blood and seminal homeostasis (154;208;211;265;266). More recently, a number of well-
known components of the blood coagulation and fibrinolysis systems, including PCI,
PUTATIVE KLK14-MEDIATED CASCADES 79 tissue- and urokinase type plasminogen activator, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and blood coagulation factor X (FX), have been identified in seminal plasma (267-271), raising the possibility of a similar proteolytic cascades in this fluid.
Here, for the first time, we propose a potential cascade-mediated role of KLK14 upstream of multiple KLK members. KLK14 is considered as the key trypsin-like protease in the SC of skin, involved in corneocyte shedding (67). Even though its downstream targets in skin are not fully understood, previous reports have implicated
KLK14 in skin proteolytic cascades. Moreover, given the significant overlap between proteins expressed in skin and seminal plasma, KLK14 could be a strong candidate regulatory protease in seminal plasma.
Our in-vitro data indicate that KLK1, 3, and 11 are regulated by KLK14.
Activation of proKLK3 is of particular interest due to its restricted expression and functional importance in seminal plasma. KLK3 is activated by cleavage after arginine at position 7 (272). Given its chymotrypsin-like substrate preference, this would exclude the possibility of autoactivation. Thus far, several trypsin-like KLKs have been reported as potential activators of proKLK3. KLK2 was initially reported as the main activator of proKLK3 (115;156;273). However, subsequent reports implied that active KLK2 is unable to cleave the propeptide sequence of KLK3(155), calling into question the previous finding. Additional prostatic KLKs, including KLK4 and 5 have also been identified as potential proKLK3 activators (115;154).
Our data suggest that KLK14 regulates the activity of KLK3 bidirectionally.
Activation occurs within a few minutes and continues up to 30 minutes. Subsequent
PUTATIVE KLK14-MEDIATED CASCADES 80
deactivation possibly occurs through internal cleavage of active KLK3. Internal cleavage
and subsequent degradation is one of the key mechanisms responsible for KLK3
inactivation. Purified KLK3 from seminal plasma contains fragments cleaved between residues Arg (85)- Phe (86), Lys(145)- Lys(146), and Lys (182)- Ser(183) (274-277). Our
previous work identified KLK5- mediated fragmentation at positions 85 and 182 (154).
However, the enzyme responsible for cleavage after lysine 145 was unknown until now.
Here, we have shown that KLK14 catalyzes the cleavage and inactivation of KLK3 at
this site.
In addition, we demonstrated that KLK14 is able to activate proKLK11. Even
though not fully characterized, KLK11 is one of the two most highly expressed KLKs in
the SC of skin (65;162). Similarly, the concentration of KLK11 in seminal plasma ranks
third after KLK3 and KLK2 (57;162). Seminal plasma depleted from KLK11 has
previously been shown to retain its ability to cleave and activate proKLK11 in vitro(57),
suggesting that seminal plasma contains KLK11 activator enzyme(s). Despite the fact
that KLK2 and plasmin had initially been identified as candidate activators of this
enzyme, further experiments ruled out activation via KLK2 (57). Here, we identified
KLK14 as an upstream activator of proKLK11, functioning as quickly as 2 minutes, at the physiologically relevant molar ratio of 1:10.
Despite the fact that KLK1 is known for over 50 years, its physiological activating enzyme remains elusive. Yet, emerging evidence points to a possible cascade- mediated function of the protein. For instance, in skin, KLK1 has been implicated in SC desquamation, through cleavage of DSG1 (66). Given the substrate overlap between
KLK1 and other KLKs of the skin proteolytic cascade, it is conceivable that KLK1
PUTATIVE KLK14-MEDIATED CASCADES 81
functions through a common proteolytic network. In seminal plasma, along with other
seminal KLKs, KLK1 is secreted from the prostate gland and is known to complex with
PCI (181;278). PCI has been shown to complex with several other seminal KLKs, including the two prominent members of the proteolytic cascade, KLK2 and 3 (179;181).
Clinically, prostatic KLK1 has been associated with insufficient sperm motility, underlying a male subfertility condition described as asthenospermia (279). Sperm motility is reportedly improved in these patients upon KLK1 administration (280). At present, kinin is the only recognized terminal inducer of sperm motility (281). Active kinins are released from seminal kininogens through limited proteolysis by a number of kininogenases, including KLK1 (116;148). Sperm motility is believed to be mediated through the B2 subtype of bradykinin (B2R) receptor and subsequent release of intracellular Ca2+ in testicular peritubular cells (280;282). However, kinin antagonists
failed to completely inhibit sperm motility (283), suggesting an alternative mechanism
whereby sperm motility is stimulated independent of the kinin signaling pathway.
Impaired sperm motility may be caused by a number of other conditions, including
incomplete, delayed, or non liquefaction of semen (208-210). Whether KLK1 partially
affects sperm motility through the liquefaction cascade needs to be further explored.
Here, we propose for the first time that KLK14 is a potential candidate activator
of KLK1. However, the recombinant proKLK1 produced in the mammalian expression
system was partially active in the absence of KLK14, suggesting its autoactivation or
proteolytic activation by other proteases. Since the KLK1- stably transfected HEK293 cell line is devoid of any other KLK expression, additional protease families may be involved. As mentioned previously, proteases with trypsin-like activity can potentially
PUTATIVE KLK14-MEDIATED CASCADES 82
function as KLK activators. We have begun to investigate alternative KLK1 activation mechanism(s) through possible cross-talks, using various approaches such as the activity based protein profiling and multidimensional protein identification technologies
(MudPIT) (284;285).
In conclusion, the data presented here strongly suggest an additional level of complexity to the modeled proteolytic cascades in skin and seminal plasma (Fig. 2.6 and
2.7). Even though trigger factors of these cascades remain to be fully elucidated, skin desquamation may be stimulated by SC acidification and subsequent release of active initiator KLK5 (286). In seminal plasma, cascade activation is more likely triggered at the time of semen ejaculation due to an immediate drop in the available Zn2+, as this ion
is spontaneously chelated by Sg proteins (220-224).
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CHAPTER 3 Validation of the Putative KLK14- Mediated Cascade in Seminal Plasma
Sections of this chapter were published in the Journal of Biological Chemistry:
Emami N, Deperthes D, Malm J, and Diamandis EP. Major role of human KLK14 in seminal clot liquefaction J Biol Chem. 2008. 283(28):19561-9.
Copyright permission has been granted.
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3.1. INTRODUCTION
Our recent work implicates KLK14 as a potential activator of proKLK3 as well as
several other seminal proKLKs, i.e. proKLK1, and proKLK11 (287). Characteristic to
classic proteolytic cascades, we have previously proposed a bidirectional regulatory
mechanism of KLK3, in which KLK14- mediated activation is followed by inactivation
via internal cleavage of active KLK3 at position Lys (145) (287).
Given the efficient inhibition of KLK14 by zinc ions and the major role of its
potential downstream target, i.e. KLK3, in semen liquefaction, it is conceivable that
KLK14 is directly or indirectly involved in this highly temporally regulated process. In
an attempt to delineate a possible function of KLK14 in seminal plasma, this study
examines the interaction between this enzyme and other potential components of the
seminal proteolytic cascade involved in semen liquefaction.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 85
3.2. EXPERIMENTAL PROCEDURES
3.2.1. Reagents
The synthetic substrates, Suc-Arg-Pro-Tyr-pNA.HCl (RPY-pNA) and Pro-Phe-
Arg-AMC (PFR-AMC)/ Gln-Ala-Arg-AMC (QAR-AMC) were purchased from
BACHEM (King of Prussia, PA) and Pharmacia Hepar-Chromogenix (Franklin, OH,
USA), respectively. Recombinant proKLK3 produced in E.Coli, was a gift from Spectral
Diagnostic Inc (Toronto, ON, Canada). Mouse anti-KLK3 monoclonal antibody was
purchased from Medix MAB (Kauniainen, Finland). Recombinant KLK1, 4, 5, 11, 12,
and 14, KLK14-specific monoclonal (clone 2E9), and rabbit anti-KLK14/KLK3
polyclonal sera were produced in house, as described previously (189). Recombinant
KLK2 was a gift from Hybritech Inc. (San Diego, USA). Conjugated goat anti-rabbit
antibody and chemiluminescent substrate for western blot were purchased from Jackson
Immunoresearch Laboratories, PA, USA and Diagnostic Products Corp., CA, USA,
respectively. NHS-activated Sepharose 4 Fast Flow beads were purchased from GE
healthcare (Pittsburgh, USA). HUK-IgG antibody recognizing KLK1 and purified Sg
proteins were kindly provided by Prof. J. Chao (Medical University of South Carolina,
USA) and Dr. J. Malm (Malmö University Hospital, Sweden), respectively. ACTG9, a
KLK14- specific recombinant mutant inhibitor was developed in collaboration with Dr.
D. Deperthes (Med-Discovery, Switzerland) by replacing the scissile bond of the reactive center loop (RCL) of α1- antichymotrypsin (ACT) inhibitor with KLK14 phage display-
selected G9 (TVDYA) substrate, as described in detail elsewhere (40).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 86
3.2.2. Materials
Coagulated semen, having a normal liquefaction rate at room temperature, was collected, split into three fractions and frozen immediately after ejaculation in liquid nitrogen. Samples were stored at -80°C until required. Liquefied semen was obtained from 95 subjects with normal and delayed liquefaction, under informed consent and approval by the Institutional Review Boards of Mount Sinai Hospital and the University
Health Network (UHN). If required, semen coagula were artificially emulsified, by addition of a small amount of chymotrypsin enzyme at 37°C for up to 1 hour.
3.2.3. Enzyme-Linked Immunosorbent Assay (ELISA)
Expression level of KLK14 protein was measured using a sandwich type ELISA, with a mouse monoclonal/rabbit polyclonal configuration, as described previously
(189;288). Briefly, 500ng/well of the monoclonal antibody against KLK14 (clone 2E9), diluted in coating buffer [50mmol/L Tris, 0.05% sodium azide (pH 7.8)], was immobilized on a 96-well white polystyrene plate overnight at room temperature. The plate was subsequently washed 2 times with washing buffer [50 mmol/L Tris, 150 mmol/L NaCl, 0.05% Tween 20 (pH 7.8)].
Seminal plasma samples were diluted 1:10 in assay buffer [50 mmol/L Tris, 6% bovine serum albumin, 10% goat IgG, 2% mouse IgG, 1% bovine IgG, 0.5 mol/L KCl,
0.05% sodium azide, pH 7.8] and were loaded and incubated for 2 hours with shaking at room temperature. The plate was then washed 6 times. 100 µl of rabbit anti-KLK14 polyclonal sera, diluted 1000-fold in assay buffer, were added and incubated for 1 hour.
The plate was washed 6 times and incubated with alkaline phosphatase (ALP)-conjugated goat anti-rabbit IgG (3000- fold dilution) for 45 minutes. Finally, diflunisal phosphate
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 87
(100µL of a 1mM solution) in substrate buffer (0.1 mol/L Tris, pH 9.1, 0.1 mol/L NaCl, and 1 mmol/L MgCl2) was added to each well and incubated for 10 minutes followed by
addition of developing solution (100 µL, containing 1 mol/L Tris base, 0.4 mol/L NaOH,
2 mmol/L TbCl3, and 3 mmol/L EDTA) for 1 minute. The resultant fluorescence was measured with a time-resolved fluorometer (Envision, Perkin- Elmer Corp. Waltham,
MA). Similarly, expression level of KLKs 1, 2, 4, 5, 11, and 12 was measured using highly specific ELISA assays, developed in-house and described previously (289).
3.2.4. Measurement of Clinical Parameters of Semen
Liquefaction rate was estimated by attempting to draw the specimen into a Pasteur pipette. Complete liquefaction is achieved when all the fluid entered the pipette. In addition, liquefaction level was evaluated visually by a phase-contrast microscope, as a measure of disappearance of the gel-like coagulum structure. Overall sperm motility (%) was determined using automated computer-assisted semen analysis (CASA) as (a+b/
(a+b+c))×100, where “a”, “b”, and “c” represent number of progressively motile sperm, sperms moving in random directions, and non-motile sperms, respectively. Cases with % sperm motility of equal or less than 35% were considered as asthenospermic.
3.2.5. Cleavage of Sg I and II Proteins
500 ng of purified SgI and SgII were incubated individually with 56 ng of KLK14 in 30 µl of KLK14 optimal assay buffer [100mM phosphate buffer, 0.01% Tween 20, pH
8.0] at 37°C for various time points. Reactions were snap-frozen in liquid nitrogen and run on SDS-PAGE gels under reducing conditions. Gels were silver-stained to visualize fragmentation.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 88
3.2.6. Sg- Mediated Reversal of Zn2+ Inhibition
To examine Sg-mediated reversal of Zn2+ inhibition, 12nM of KLK14 was
incubated with 0 and 120 nM of Zn2+ (in the form of zinc acetate), at a final volume of
100µl, for 10 minutes at 37°C. Subsequently, the fluorogenic substrate QAR-AMC was added at the final concentration of 1mM. Fluorescence release was measured on a Wallac
Victor fluorometer (Perkin- Elmer Life Sciences), set at 355 nm for excitation and 460 nm for emission. Fluorescence was measured for a total of 20 minutes. Five minutes after initiating the read, 0.05µM of each SgI, SgII, or 0.01M EDTA was added to each well.
Measurement was resumed as described above. Background fluorescence was subtracted from raw values. All experiments were performed in triplicate.
3.2.7. Enzyme Activity Assays
The “chymotrypsin-like” activity of seminal plasma samples (diluted 10 times) was kinetically examined, using 0.8 mM of the colorimetric substrate RPY-pNA in a final volume of 100µl of KLK3 optimized assay buffer (0.1mM Tris, 3mM NaCl, 0.01%
Tween 20, pH 7.5). Absorbance was measured on a Wallac Victor Fluorometer at 405 nm. Background absorbance was subtracted from raw values of seminal plasma alone and samples treated with either active recombinant KLK14 or ACTG9, described above.
Reactions were repeated 3 times. KLK1-specific activity was measured by fluorescence
release of the pulled down KLK1 protein, as previously described (287). Briefly, 200 ng of KLK1-specific polyclonal antibody (HUK-IgG) were immobilized on a 96-well white polystyrene plate overnight. The plate was washed two times prior to addition of reaction mixtures. KLK1 activity was measured as an increase in the fluorescence of PFR-AMC
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 89 substrate after two hours of sample incubation. Reaction rates (fluorescence units/minute) correspond to the slope of the fluorescence release-time plot.
3.2.8. KLK3 Depletion From Seminal Plasma
1 mg of monoclonal anti-KLK3 antibody was immobilized on 1ml of 50% NHS- activated Sepharose Fast Flow bead slurry, according to the manufacture’s protocol.
Briefly, beads were equilibrated three times in 2ml of ice-cold 1mM HCl. They were then were incubated with 1mg of monoclonal anti-PSA antibody for 1 hour at room temperature with end-over-end mixing. Residual active groups of beads were subsequently blocked by washing beads sequentially three times with 2ml of each buffer
A (50mM Tris.HCl, 1M NaCl, pH 8.0) and buffer B (0.1 M acetate, 0.5M NaCl, pH 4.0).
Beads were further washed two times with buffer A and then incubated for 15 minutes at room temperature. Further blocking was achieved by sequential incubation of beads three times with each buffer B, A, and B. Beads were equilibrated for protein binding in TBS
(50mM Tris, 150mM NaCl, pH 7.5). 20µl of seminal plasma were diluted in TBS in total volume of 1ml and incubated with beads for 1 hour at room temperature, with end-over- end mixing. The flow through (depleted samples) were collected and further analyzed kinetically. Beads were washed 5 times in wash buffer (TBS with 2M urea, pH 7.5) and eluted with 1ml of elution buffer (0.1M glycine with 2M urea, pH 3.0). A mock depleted sample was prepared in parallel, using beads alone.
% depletion was estimated by measuring KLK3 in flow through samples, using
KLK3- specific ELISA. Collected flow- through samples were concentrated 10 times, using membranes with molecular weight cut-off of 5KDa. 5µl of concentrated samples were diluted in 95µl of KLK3 optimal assay buffer. Enzymatic activity towards the
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 90 tripeptide RPY-pNA substrate was measure as described above. To ensure that the observed drop of enzymatic activity is due to exclusive depletion of KLK3, two identical reactions of 20µl of immuno- and mock-depleted elutions were run on SDS-PAGE under reducing conditions. One gel was silver-stained and the other was immunoblotted with anti-KLK3 antibody as described below.
3.2.9. Western Blotting for Identification of KLK3 Fragmentation in Seminal Plasma
To monitor KLK14-mediated fragmentation of KLK3 ex-vivo, semen coagula were spiked for 1hour with various amounts of active recombinant KLK14 and were analyzed by western blot. Similarly, KLK14-mediated fragmentation of proKLK3 was re- confirmed in-vitro by incubating recombinant proKLK3 with active KLK14 at 10:1 molar ratio for varying time points, in a total volume of 30 µl. Recombinant and seminal proteins were resolved by SDS-PAGE, using the NuPAGE Bis-Tris, with 4-12% gradient polyacrylamide gels at 200 V for 45 min and transferred onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 volts for 1 hour. The membrane was subsequently blocked for 1 hour with 5% milk/TBS-Tween [0.1 mol/liter Tris- HCl containing 0.15 mol/liter NaCl and 0.1% Tween 20] at 4°C and probed using rabbit anti-
KLK3 polyclonal sera (diluted 1:1000) for 1 hour at room temperature. The membrane
was washed three times for 15 minutes with TBS-Tween and treated with ALP- conjugated goat anti-rabbit antibody (diluted 1:8000) for 45 minutes at room temperature.
The membrane was re-washed as above, and fluorescence was detected on X-ray film using a chemiluminescent substrate.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 91
3.3. RESULUTS
3.3.1. Clinical Association Between KLK14 Expression and Liquefaction Rate
KLK14 concentration in seminal plasma from 95 volunteers (including 34 normal cases and 61 patients with delayed liquefaction) ranged from 0.2 to 181.2 µg/L, with a mean of 13.2 µg/L and a median of 6.8 µg/L. The expression level of KLK14 had a
median of 5.2 µg/L and 11.55 µg/L and mean of 12.38 µg/L and 12.99 µg/L in samples with delayed and normal liquefaction, respectively (Fig. 3.1A). We concluded that
KLK14 levels were significantly decreased (p= 0.0252) in the patient group with delayed liquefaction.
In addition, KLK14 expression was found to be significantly (p= 0.0478) lower in
70 asthenospermic patients (15 cases with undetermined or inconclusive % motility were excluded from the study) (Fig. 3.1B). The level of KLK14 was dropped to 9.8 µg/L
(mean) and 7.9 (median) in asthenospermic cases, as compared to normal individuals with the mean value of 22.5 µg/L and the median of 13.4 µg/L.
3.3.2. Role of KLK14 As a Seminal Liquefying Protease
To further investigate the possible role of KLK14 in semen liquefaction, the
proteolytic activity of the enzyme in seminal plasma was induced, and reciprocally
inhibited, by using either active recombinant KLK14 or the highly specific KLK14
inhibitor ACTG9, respectively. Complete liquefaction ranged from 10 to 20 minutes in
normal samples. Addition of ACTG9 inhibitor to a split fraction of a normal ejaculate
sample strongly delayed liquefaction (≥ 30 minutes). As expected, the progression of
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 92 liquefaction was also reduced in inhibitor- treated samples, as the gel-like coagulum structure persisted longer than their untreated control counterparts (Fig. 3.2).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 93
A). B).
FIGURE 3.1. Clinical association between KLK14 expression and A). liquefaction rate. Distribution of KLK14 concentration (µg/L) in liquefied seminal plasma of healthy males (normal) and individuals with delayed liquefaction (i.e. complete liquefaction did not occur naturally up to 45 minutes post- ejaculation). B). asthenospermia. Individuals with ≤ 35% sperm motility were considered as clinically asthenospermic. The p value was determined by the Mann-Whitney, t-test. Horizontal lines represent the median values.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 94
A). B). C).
D). E). F).
FIGURE 3.2. Optical analysis of liquefaction level of semen coagulum. The general gel-like structure of the clot is visible under a phase contrast microscope, with sperms (arrowheads) entrapped in its cavities. Each of the three splits of a same ejaculate was treated with (A, D). KLK14- specific inhibitor. KLK14 activity was specifically inhibited using 0.8 µM of the recombinant mutant serpin ACTG9. (B, E). Distilled water, a control. (C, F). Recombinant KLK14. The sample was spiked with 0.8 µM of the recombinant active KLK14. Liquefaction level was estimated, as a measure of disappearance of semen coagula. Note the gradual decrease in the intensity of the coagulum structure after 10 minutes of incubation at room temperature. Scale bars: 4µm (A, B, C) and 10µm (D, E, F).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 95
Conversely, liquefaction was accelerated upon addition of active recombinant
KLK14. The gel-like structure of semen coagula seemed to be less dense in KLK14- induced samples (Fig. 3.2). The coagula of normal liquefying ejaculates were dissolved too fast; for this reason, we could not determine the effect of KLK14 on liquefaction rate.
3.3.3. Cleavage of Sg Proteins by KLK14
Given the pronounced effect of KLK14 on semen liquefaction, we next examined whether any of the primary components of semen coagulum function as immediate downstream targets of KLK14. The ability of KLK14 to cleave purified SgI and II proteins was tested. SgI and II were incubated with active recombinant KLK14 in separate reactions. KLK14 was able to almost fully cleave both SgI and II, as quickly as
12 minutes of incubation (Fig. 3.3). New fragments were generated as early as 2 minutes after initiation of the reaction.
3.3.4. Reversal of Zn2+ Inhibition by Sg I and II
Zn2+ has previously been proposed to function as a cationic protease inhibitor of
KLK14 (189). As mentioned previously, SgI and II can indirectly regulate the activity of
a number of KLKs by binding to Zn2+ molecules, rendering them unable to inhibit KLK activity. To examine whether Sg proteins have the same effect on Zn2+- mediated
inhibition of KLK14, KLK14 was incubated with 10 times molar excess of Zn2+. The
enzymatic activity of KLK14 was monitored kinetically as above. Addition of SgII after
5 minutes of initiation of the reaction rapidly reversed the inhibition (Fig. 3.4), suggesting
a common regulatory mechanism with several other seminal KLKs. No such effect was
observed for SgI (data not shown).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 96
FIGURE 3.3. KLK14- mediated degradation of semenogelin proteins. KLK14 (56 ng) was incubated with 500 ng of purified Sg I and Sg II for varying time intervals. The mixtures were resolved by SDS-PAGE under reducing conditions and the gel was sliver- stained. Major KLK14- generated fragments of SgI and SgII are indicated by stars and arrowheads, respectively. M, molecular mass standards in KDa.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 97
FIGURE 3.4. Reversal of Zn2+ inhibition by semenogelin II. Cleavage of QAR-AMC by KLK14 (12nM) in presence of: optimal assay buffer only, 0.01M EDTA, 0.05 µM of Sg II, 120 nM Zn2+ , and 120 nM Zn2+ plus 0.01M EDTA or 0.05µM of Sg II. The arrow (↓) shows time of addition of EDTA or Sg II. Note the increase of the residual activity of Zn2+- inhibited KLK14 to almost basal level after addition of Sg II.
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3.3.5. Correlation Between KLK14 and the “Chymotrypsin-Like” Activity
ProKLK3 was previously proposed to function downstream of KLK14, in-vitro
(287). Unfortunately, there is no tool currently available to specifically quantitate KLK3 enzymatic activity in complex biological samples such as seminal plasma. However, according to our screening of tripeptide substrates, KLK3 shows preference to substrates with P1- tyrosine, P2-proline, and P3- arginine (unpublished data). Given that KLK3 is the major chymotrypsin-like enzyme in seminal plasma and its preferential substrate recognition, we reasoned that KLK3 activity could accurately be estimated in seminal plasma by measuring the chymotrypsin activity towards the RPY-pNA substrate. To corroborate this assumption, a series of ex-vivo depletion experiments were performed.
Seminal plasma with approximately 95% depleted KLK3 exhibited almost zero activity towards the RPY tripeptide substrate, as compared to the mock depleted control (Fig.
3.5A). In addition, eluted samples of depleted and mock controls were examined by silver stain and western blotting against KLK3 (Fig.3.5B). All the proteins eluted from the immuno-depleted sample were successfully identified as full-length KLK3 or KLK3 fragments by western blotting, verifying the specificity of pull down.
Given that KLK3 activity could confidently be assessed by measuring the chymotrypsin-like activity against the tripeptide RPY-pNA (referred to as
“chymotrypsin-like” for short, here), KLK14-mediated regulation of KLK3 was next examined.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 99
FIGURE 3.5. KLK3 depletion of seminal plasma. 20µl of seminal plasma was depleted from KLK3, using 1mg of monoclonal anti-KLK3 antibody immobilized to NHS- activated Sepharose Fast Flow beads. Beads alone were used as control. % depletion was estimated by measuring KLK3 in flow through samples, using KLK3- specific ELISA. A). Kinetic analysis of depleted samples. Depleted samples (flow throughs) were collected and further analyzed kinetically. B). Silver staining and C). Western blot analysis of eluted proteins. Beads were washed and eluted. The immuno- and mock- eluted fragments were separated on SDS-PAGE gels and silver stained. KLK3-related fragments were identified using western blot analysis against KLK3. Note the complete overlap between fragments identified by silver staining and western blotting, confirming the specificity of the pull down.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 100
The “chymotypsin-like” activity of seminal plasma was dependent on the level of
KLK14 activity, since samples treated with active recombinant KLK14 exhibited
approximately 78% higher “early” (30 minutes after ejaculation) “chymotrypsin-like”
activity, compared to those treated with the KLK14 inhibitor (Fig. 3.6A). As previously
suggested, the observed increase was rapid and transient, followed by a decrease in the
“chymotrypsin-like” activity. The reaction rate declined following longer incubation (90
minutes post-ejaculation) of seminal coagula, resulting in a reversal of the activity pattern
of treated samples vs. the untreated controls (Fig. 3.6B). The “chymotrypsin-like”activity
of ACTG9- treated samples increased approximately 10%, while a drop of almost 78%
was seen in samples spiked with active recombinant KLK14 (Fig. 3.6B).
3.3.6. Fragmentation of Seminal KLK3 by KLK14
Our previous in-vitro work suggests an inactivation mechanism of KLK3 through
internal cleavage of the active protein. To confirm this, we compared degraded products
of KLK3 in-vitro and in seminal plasma by western blotting, using rabbit anti-KLK3
polyclonal sera. All major fragments identified previously by silver staining (287) were detected by our antibody (Fig. 3.7A). A very similar fragmentation pattern was observed in seminal plasma spiked with various amounts of active recombinant KLK14
(Fig. 3.7B). As expected, fragmentation was dependent on the level of KLK14 activity.
Interestingly, the prominent band generated following KLK14 induction has the molecular mass of the previously identified fragment produced uniquely by KLK14, after cleavage of KLK3 at the peptide bond Lys (145)-Lys (146) (287).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 101
A).
B).
FIGURE 3.6. Regulation of total chymotrypsin activity by KLK14. Ejaculate splits were incubated alone, or were individually treated with 0.8µM of active recombinant KLK14 or KLK14 inhibitor ACTG9, prior to the incubation. Treated and control samples were incubated at room temperature for A). Early total chymotrypsin activity. (30 minutes) B). Late total chymotrypsin activity. (90 minutes). Total chymotrypsin activity was monitored by cleavage of the RPY-pNA substrate(0.8µM). Residual reaction rates of the treated samples were normalized to the basal reaction rate of the untreated sample. Note the increase of chymotrypsin activity at 30 minutes, after addition of KLK14, and the subsequent decrease, 90 minutes after treatment. The chymotrypsin activity of the sample treated with ACTG9 inhibitor was slightly elevated at 90 minutes.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 102
A).
B).
FIGURE 3.7. KLK14- mediated internal cleavage of A). Recombinant KLK3. Pro-KLK3 was incubated with KLK14 at a 1:10 molar ratio for varying time intervals, at 37°C. B). Seminal KLK3 ex- vivo. 2µl of seminal plasma, containing approximately 22nM of total KLK3, were diluted 15 times in PBS and treated with either 0.3µM ( 1:70 molar ratio) or 0.7 µM (1:30 molar ratio) of KLK14. Reaction mixtures were incubated for 1 hour at room temperature. KLK3 fragments were immunodetected, using a rabbit polyclonal KLK3 antibody (1:1000). Filled arrowhead represents fragments generated from the recombinant KLK3 but not detected in B. Open arrowheads illustrate common fragments to the recombinant and seminal KLK3. The asterisk shows a dose-dependent increase in the intensity of one of the KLK3 fragments in seminal plasma. SP, seminal plasma.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 103
3.3.7. Activation of Seminal KLK1 by KLK14
KLK1 has been proposed as one of the downstream targets of KLK14, in- vitro
(287). To evaluate possible KLK14-mediated activation mechanism of seminal KLK1, we examined KLK1-specific activity in ACTG9 treated samples as compared to an untreated split fraction of the same ejaculate. The specific activity of KLK1 was attenuated approximately 20% upon treatment of the ejaculate with the ACTG9 synthetic inhibor against KLK14 (Fig. 3.8A). This would support that KLK14 could activate pro-
KLK1 in seminal plasma.
Given the high abundance of trypsin- like KLKs with overlapping substrate specificity, it is critical to ensure pull-down specificity of the KLK1 antibody. In order to exclude the possibility of nonspecific pull down of physiologically relevant KLKs, protein expressions of KLK1, 2, 4, 5, 11, 12, and 14 were measured using ELISA assays developed in-house (Table 3.1). The pull-down specificity of anti- KLK1 HUK IgG was evaluated using active recombinant KLK2, 4, 5, 11, 12, and 14 in their equivalent amounts found in seminal plasma (Fig. 3.8B). Although these KLKs are highly active when soluble (data not shown), almost no enzymatic activity was observed after they were pulled down with KLK1 antibody.
Based on the information provided above, a novel cascade pathway for KLK14 function in semen liquefaction was developed (Fig.3.9).
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 104
Table 3.1. Expression level of trypsin-like KLKs in seminal plasma KLK Expression Level (nM)* KLK1 1.7 KLK2 93 KLK4 0.4 KLK5 0.08 KLK11 163 KLK12 0.2 KLK14 0.13 * An approximate molecular mass of 30 KDa has been used for all KLKs. All data were generated by specific KLK ELISA assays.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 105
A).
B).
FIGURE 3.8. KLK14- mediated activation of seminal KLK1 A). Ex-vivo activation. Split ejaculates were treated with 0.8µM of the ACTG9 inhibitor or incubated alone. Samples were incubated at 37°C for 10 minutes. KLK1 was pulled-down in 96 microtiter plates, coated anti-KLK1 antibody, as follows: 200 ng of anti-KLK1 antibody were immobilized overnight on a microtiter plate. 100µl of each of the treated and untreated samples were loaded to each well, in triplicates, and incubated at room temperature for 2 hours. Activity of the pulled-down KLK1 was monitored by cleavage of 0.5mM of the PFR-AMC substrate. B). Specificity of the KLK1 sandwich pull down assay. Recombinant active KLK1, 2, 4, 5, 11, 12, and 14 were loaded on a KLK1- antibody coated microtiter plate, at their physiologic level listed in table 3.1.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 106
FIGURE 3.9. Schematic presentation of proposed KLK cascade in seminal plasma. KLK14 activates proKLK3 as well as proKLK11 and KLK1. Activated KLK3 acts as the main executor protease in the liquefaction of semen coagulum through proteolytic fragmentation of SgI/SgII and FN. Activated KLK11 may also activate proKLK3, functioning at the propagation level. Moreover, active KLK14 can directly cleave gel-like proteins, i.e. SgI/SgII and FN. Signal amplification is achieved mainly through positive feedback loops. The cascade is regulated by a number of endogenous inhibitors shown as “Inh”, as well as Zn2+, and internal cleavage of active KLKs. Solid lines specify interactions that were confirmed ex-vivo in this study. Dotted lines represent those that have been shown in-vitro, using full-length recombinant proteins. The question marks indicate possible interactions suggested in-vitro, using fusion recombinant proteins that contain only the active motifs of each KLKs.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 107
3.4. DISCUSSION
Human semen coagulates spontaneously after ejaculation and consequently
liquefies within 5-20 minutes under normal physiological conditions (212). Although the
mechanism is not fully understood, the process of semen coagulation/ liquefaction is believed to be regulated through a series of enzymes, mainly proteases, and inhibitory
factors (215;216).
More recently, a number of well-known components of the blood coagulation and
fibrinolysis systems have been identified in seminal plasma and were associated with
male fertility (267-271). Given the overlapping regulatory components of the seminal and
blood homeostasis, this emerging evidence suggests that analogous to fibrinolysis, semen
liquefaction is regulated through highly orchestrated proteolytic cascades
(154;208;211;265;266).
Accumulating evidence suggests that several members of the KLK family
participate in the seminal proteolytic cascade and are involved in the process of
degradation of the semen coagulum (154). In-vitro data by our group and others suggest
that KLK14 might function as a key factor in the proteolytic cascade in seminal plasma,
regulating major seminal KLKs, including KLK1, KLK3 and KLK11 (287;290).
Furthermore, the enzymatic activity of KLK14 has recently been shown to be inhibited
by Zn2+ (189), strengthening the proposed function of the enzyme in seminal plasma and prostatic tissue.
Here, for the first time, we propose a cascade-mediated role for KLK14 in seminal plasma, as one of the key trypsin-like regulatory proteases involved in liquefaction of the seminal coagulum.
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 108
Trypsin-like proteases are of main importance, as they can function as activators of KLKs that are unable to self-activate (194). A prime example of KLKs lacking auto- proteolytic ability is the chymotrypsin-like enzyme KLK3. As mentioned previously,
KLK3 has extensively been studied as a main executor KLK in seminal plasma, functioning through cleavage of gel-like proteins and initiating semen liquefaction
(161;223). However, surprisingly, no significant difference was found in KLK3 expression level between normal and delayed-liquefaction (291), suggesting possible aberration at the regulatory level of the protein, due to insufficient activation. Previously, we reported KLK14 as an activator of proKLK3. Interestingly, our clinical data indicate that there is a significant correlation between abnormal liquefaction and asthenospermia vs. the expression level of seminal KLK14. The physical constraint of retained coagula seems to adversely affect sperm motility, as we observed approximately 70% drop in number of motile sperms in samples with delayed liquefaction (data not shown). Whether the observed reduced level of KLK14 is due to its abrogated expression in the prostate or its partially obstructed secretion to seminal plasma remains to be determined.
In addition, using targeted inhibition and reciprocal over-activation of KLK14 in seminal plasma, we demonstrated that KLK14 is vital for complete liquefaction of the seminal clot. The mutant inhibitor ACTG9 used in this study is highly potent and selective towards KLK14 (40). ACTG9 contains mutations at the RCL of the biological inhibitor
ACT, converting the natural RCL to the phage display- selected KLK14 substrate G9
(40). This would confer an excellent inhibitory specificity towards KLK14; other major seminal KLKs, including KLK2, 3,4, 5, and 12 were not inhibited by this protein (40) and our unpublished data).
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Even though KLK14 inhibition considerably delayed semen liquefaction, it did not completely block the process. This suggests functional redundancy in activator components of the seminal proteolytic cascade, compensating for KLK14 function. The physiological relevance of other candidate activators of the cascade, such as KLK5 and
KLK2, needs to be further investigated.
As mentioned previously, Sgs are the main effector components of the semen liquefaction cascade. Our in-vitro data indicate that KLK14 cleaves Sg proteins with high efficiency. In addition, our previous studies have implicated KLK14 in the processing of
FN, another key component of the semen coagulum (223). Furthermore, Sg proteins play an instrumental role in seminal clot liquefaction through sequestration of Zn2+ from active executors, thus modulating their proteolytic activity (227). Such reversal effect of
Sg has been shown for several members of the KLK family, including KLK3 and KLK5
(154;161). Our results suggest a similar regulatory mechanism for KLK14 in seminal plasma, at the physiologically relevant molar ratio of 10-fold excess Zn2+ (161) to SgII protein.
Moreover, we previously demonstrated that KLK14 is able to regulate proKLK3, in-vitro. At the astounding expression level of 10 mg/ml, KLK3 is the most abundant chymotrysin-like enzyme in seminal plasma (272;292). However, the majority of active
KLK3 is complexed with seminal inhibitors such as PCI and α2- M (160;293;294), rendering it inactive. While a number of chromatographic and immunologic approaches have previously been proposed to measure active KLK3 (274;295;296), their low recovery rate limits their use as a sensitive comparative means in complex biological samples. Due to this technical limitation and given the substrate preference of KLK3 to
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 110 the RPY tripeptide substrate, we examined the “chymotrypsin-like” activity of seminal plasma as a measure of KLK3 activity. To ensure that the majority of observed enzymatic activity against this substrate is due to KLK3 activity, we compared samples depleted from KLK3 with mock controls. As expected, upon 95% depletion of KLK3, almost no enzymatic activity was observed. Eluted proteins were identified as KLK3 or KLK3 fragments, excluding the possibility of simultaneous depletion of other chymotrypsin-like enzymes.
KLK14- mediated regulation of KLK3 activity seems to be bidirectional, as we observed a reversal in the correlation pattern between KLK14 and the “chymotrypsin- like” activity following longer incubation. Given the importance of chymotryptic proteolysis of Sg proteins during semen liquefaction, activation of KLK3 is most likely triggered within seconds post ejaculation and continues until complete fragmentation of gel-like proteins. Aberrant proteolysis due to prolonged protease activity is prevented by subsequent inactivation of executor chymotryptic enzyme(s). This finding is in agreement with our in-vitro observation of sequential activation and deactivation of proKLK3 by
KLK14 (287). We have previously found that deactivation is achieved mainly through internal cleavage of active KLK3 (287). Here we have shown that exogenous KLK14 could fragment KLK3 ex-vivo in a dose-dependent manner, with a pattern similar to the one observed in-vitro.
Similarly, consistent with our previous in-vitro data, KLK14 seems to activate seminal KLK1. Likewise, KLK2 has recently been identified as another putative activator of proKLK1 (290), reinforcing the link between KLK1 and the seminal KLK cascade.
Even though not fully understood, KLK1 has clinically been shown to enhance sperm
KLK14-MEDIATED CASCADE IN SEMINIAL PLASMA 111 motility in asthenospermic patients (279;280). As mentioned previously, semen liquefaction is one of the main post-ejaculatory determinants of sperm motility. Whether
KLK1 functions through regulating coagulation/liquefaction of semen needs to be further explored.
In summary, the present study provides strong evidence for the crucial cascade- mediated function of KLK14 in regulating the coagulation and liquefaction of human semen. Cascade activation is more likely triggered at the time of semen ejaculation, as a result of mixing of different components of seminal plasma and subsequent redistribution of Zn 2+ to Sg proteins. It is conceivable that additional members of the KLK family and/or other proteases participate in this proteolytic cascade. In addition, the complex interplay between proteases and their regulatory check points needs to be further elucidated.
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CHAPTER 4 Association Between Seminal KLKs and Macroscopic Indicators of Semen Analysis
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4.1. INTRODUCTION
Even though the mechanism of coagulation/liquefaction of human semen is fairly well understood, etiological factors leading to abnormal liquefaction in infertile men are still largely unknown. Similarly, probable factors associated with the etiology of hyperviscous samples remain to be investigated.
As mentioned previously, several members of the KLK family have been found to be secreted in seminal plasma in varying levels. These KLKs have directly or indirectly been implicated in the process of semen liquefaction, functioning through a seminal proteolytic cascade.
In an attempt to delineate possible factors involved in the pathogenesis of delayed liquefaction and/or hyperviscosity, this study examines the expression pattern of eleven
KLKs in seminal plasma, using enzyme-linked immunosorbent assays. The clinical value of these KLKs in the evaluation of semen quality and in the differential diagnosis and etiology of abnormal liquefaction and/or viscosity is further examined.
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4.2. MATERIALS AND METHODS
4.2.1. Clinical Samples
One hundred and thirteen semen samples were obtained from the diagnostic semen laboratory at Mound Sinai Hospital between Octorber2007 and Februarys2008, under informed consent and approval of the Institutional Review Boards of Mount Sinai
Hospital and the University Health Network (UHN).
The results of CASA, including total cell concentration, number of motile sperms, straight line speed, %motility, motile sperm concentration, and additional macroscopic parameters, i.e. volume, and pH, as well as patient age, were collected prospectively.
Samples were centrifuged at 7000g for 10 minutes to separate the spermatozoa from the seminal plasma and kept frozen in aliquots at -80°C. Prior to use, aliquots were thawed overnight at 4°C.
The semen samples were divided into four clinical groups, according to their liquefaction and viscosity states. Group 1 (the normal group) consisted of samples with both normal liquefaction and viscosity. Group 2 included hyperviscous specimens but with normal liquefaction. Group 3 included specimens with delayed liquefaction but with normal viscosity. Finally group 4 included specimens presenting with both abnormal liquefaction and hyperviscosity.
The liquefaction and viscosity state of samples and sperm characteristics were defined according to the World Health Organization. Viscosity was determined using the modified pipette method by simply attempting to draw seminal plasma into a Pasteur pipette and slowly release it in a drop-wise fashion (260). The viscosity is reported
“normal” when single drops are released within a distance of 20 mm from the pipette tip
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(261). Accordingly, normal liquefaction was assigned if samples liquefied in less than 60
minutes at 37°C. Samples that did not liquefy after 120 minutes at 37°C were classified
as “delayed”.
Viscosity and coagulum were differentiated by swirling the container. Contrary to
viscous fluids, a delayed liquefying coagulum did not conform to its environment shape.
Some of the hyperviscous ejaculates or those that did not liquefy, were enzymatically
induced by addition of a small amount of chymotrypsin enzyme at 37°C for up to 1 hour.
Sperm motility was measured using automated computer-assisted semen analysis
(CASA). Cases with % sperm motility of equal or less than 35% were considered as
asthenospermic. Patient distributions by demographic and clinical characteristics are
presented in Table 4.1.
4.2.2. Enzyme-Linked Immunosorbent Assays (ELISA)
The concentration of various KLKs, i.e. KLK1-3, 5-8, 10, 11, 13, and 14, were
measured using ELISA- type assays developed in-house. In each assay two antibodies were used in “sandwich” to capture and detect the amount of the antigen of interest.
Three types of configurations of ELISA were used in this study, i.e. a). monoclonal- monoclonal for KLK5, 6, 7, 8, 10, and 13, b). monoclonal-polyclonal for KLK11 and 14, and c). polyclonal-polyclonal for KLK1 (Table 4.2).
The assays were standardized using recombinant proteins produced in yeast or mammalian expression systems. Assay precision within the dynamic range was estimated to be less than 10% and no cross-reactivity with other members of the family was detected for the above mentioned ELISAs. For more detailed information on the ELISAs used, see elsewhere (297).
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Table 4.1. Descriptive statistics of patient age, semen volume, sperm counts and sperm concentration in the four clinical groups
Variable Normal Normal Delayed Delayed Liquefaction, Liquefaction, Liquefaction, Liquefaction, Normal Hyperviscous(2) Normal Hyperviscous(4) Viscosity(1) Viscosity(3) Age (years) N 30 28 20 35 Mean±SE 37.5±0.8 38.0±0.9 36.7±1.1 36.4±0.9 Median 37.0 38.5 37.5 37.0
Volume (mL) N 30 28 20 35 Mean±SE 2.79±0.24 2.38±0.17 3.22±0.61 3.19±0.24 Median 2.50 2.50 2.50 3.00
Sperm counts (106) 30 28 20 35 N 920±50 957±60 894±86 865±69 Mean±SE 890 1000 891 853 Median
Sperm concentration (106/mL) 30 28 20 35 N 67.8±10.2 65.9±9.3 56.9±12.8 67.4±14.1 Mean±SE 46.2 56.3 36.1 48.8 Median
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Table 4.2. Antibodies used in the ELISA assays
Assay Capture (code) Detection (code) Source
KLK1 AP-polyclonal1 AP-polyclonal Julie Chao, MUSC KLK2 monoclonal (HK1G 586.1 ) monoclonal (8311) Beckman Coulter Inc KLK3 monoclonal (8301) monoclonal (8311) Medix Biochemica KLK4 monoclonal (10F4-1G6) polyclonal in-house KLK5 monoclonal (2A4) monoclonal in house; R&D KLK6 monoclonal (27-4) monoclonal E24 in house KLK 7 monoclonal (73-1) monoclonal (8301) in house KLK8 monoclonal (19-10) monoclonal (20-64) in house KLK9 monoclonal (M1G1-E11) polyclonal in house KLK10 monoclonal (B14) monoclonal (5D3) in house KLK11 monoclonal (18-1) polyclonal in house KLK12 monoclonal (4F3) polyclonal in house KLK13 monoclonal (11C1) monoclonal (27-1) in house KLK14 monoclonal (2E9) polyclonal in house KLK15 monoclonal (820) polyclonal in house; R&D
1 AP, affinity-purified
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4.2.3. Statistical Analysis
Since the distributions of KLKs levels were not Gaussian, the analyses of the differences between these parameters, in two or three groups, were conducted with the
Mann-Whitney and Kruskal-Wallis, respectively. Spearman’s rank correlation was used to assess the correlation among measured KLKs themselves and with other studied continuous variables in seminal plasma. To further investigate the discriminatory value of the significant KLKs in semen viscosity and liquefaction, multivariate logistic regression models were developed, adjusted only for KLKs. The log likelihood scores were calculated for these multivariate logistic regression models. The statistically significant
KLK variables for each patient were included. Regression coefficients were calculated on log-transformed KLKs. All analyses were performed using SAS 9.1 software (SAS
Institute, Inc.).
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4.3. RESULTS
4.3.1. Distribution of KLKs Among the Four Clinical Groups
To determine whether there is any clinical association between the seminal KLKs
and semen liquefaction and viscosity, the concentration of these KLKs was measured in
seminal plasma using in-house developed ELISA assays.
The concentration of KLK2, 3, 13, and 14 was significantly reduced (p=0.023,
0.008, 0.019, and 0.048, respectively) in individuals presenting with abnormal
liquefaction with or without hyperviscosity (Table 4.3). Upon further analysis, we found
that the concentrations of KLK2 and 13 declined significantly (p= 0.047 and 0.037,
respectively) in individuals with both abnormal liquefaction and hyperviscous semen,
while KLK3 concentration was significantly (p=0.038) reduced only in samples with
abnormal liquefaction and normal viscosity (Fig. 4.1). Interestingly, the concentration of
several other KLKs (i.e. KLK5, 6, 7, 8, and 10) was significantly lower (p= 0.013, 0.003,
0.034, 0.001, respectively) only when the two clinical groups with normal liquefaction and different viscosity state were compared (Fig. 4.1). No significant difference was observed in the concentration of KLK11 among the four studied clinical groups.
The correlations between measured KLKs were determined using Spearman’s rank correlation coefficients (Table 4.4). A strong positive correlation was observed among KLK5 and KLK6, 7, 8, and 10, KLK6 and KLK7, 8, and 10, and KLK2 and
KLK5 (Spearman’s rank correlations ranged from 0.648 to 0.819, p < 0.05). No correlation was found between KLK1 and KLK3, 5, 6, 7, 8, 10, 11, 13, KLK3 and KLK6,
7, 8, 10, 13, 14, and KLK7 and KLK10. The remaining KLKs exhibited weak to moderate positive correlations.
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Table 4.3. Distribution of prostatic KLKs in seminal plasma of the four clinical groups KLKs Normal Liquefaction, Normal Delayed Delayed p value* (ug/L) Normal Viscosity (1) Liquefaction, Liquefaction, Liquefaction, Hyperviscous (2) Normal Hyperviscous (4) Viscosity (3) KLK1 N 30 28 20 35 0.019a, N.Sb Mean±SE 76.6±11.8 45.5±10.5 48.6±10.8 38.2±6.2 N.Sc, N.Sd Median 66.2 28.1 36.3 23.0 KLK2 N 30 28 20 35 0.021a, N.Sb Mean±SE 29140±7200 31540±19820 20990±5540 3810±1560 0.047c, 0.023d Median 18360 6800 12240 2500 KLK3 N 30 28 20 34 N.Sa, 0.038b Mean±SE (15±2.3)x106 (6.4 ±5.1) x106 (9.0±1.5) x106 (7.6±9.0) x106 N.Sc, 0.008d Median 11x106 10x106 8x106 7x106
KLK5 N 19 20 18 31 0.013a, N.Sb Mean±SE 11.6±5.0 2.4±0.9 26.6±9.1 4.7±2.2 N.Sc, N.Sd Median 3.8 0.26 4.3 0.27 KLK6 N 28 23 18 29 0.003a, N.Sb Mean±SE 3.21±0.62 2.59±1.51 4.86±1.73 0.89±0.41 N.Sc, N.Sd Median 2.26 0.16 2.20 0.16 KLK7 N 30 28 20 35 0.003a, N.Sb Mean±SE 7.98±1.83 3.41±0.90 23.14±8.39 5.53±3.56 N.Sc, N.Sd Median 5.38 0.50 8.89 0.50 KLK8 N 27 23 18 29 0.034a, N.Sb Mean±SE 4.05±0.90 1.76±0.27 14.23±6.99 4.59±2.97 N.Sc, N.Sd Median 2.39 1.38 1.85 0.81 KLK10 N 27 24 18 29 0.001a, N.Sb Mean±SE 4.11±0.92 1.49±0.33 8.95±3.08 2.54±1.79 N.Sc, N.Sd Median 2.71 0.50 4.22 0.50 KLK11 N 30 28 20 35 N.Sa, N.Sb Mean±SE 7084±824 20214±14667 6182±1628 4562±602 N.Sc, N.Sd Median 6190.6 4528.3 4209.7 3625.4 KLK13 N 30 28 20 35 0.007a, N.Sb Mean±SE 43.2±5.0 21.1±4.3 50.9±9.9 12.5±4.2 0.037c, 0.019d Median 41.9 10.7 41.1 4.4 KLK14 N 30 28 20 35 0.004a, N.Sb Mean±SE 12.3±2.1 7.6±2.4 20.2±8.4 9.9±5.1 N.Sc, 0.048d Median 10.1 3.2 10.4 3.4 * Mann-Whitney test N.S: Non-significant (p>0.05) a Group (1) vs Group (2), b Group (1) vs Group (3), cGroup (2) vs Group (4) d Group (1+2) vs Group (3+4)
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FIGURE 4.1. Distribution of seminal plasma KLK concentrations (ug/L) in the four clinical groups. Group1: Normal liquefaction and viscosity. Group2: Hyperviscous but with normal liquefaction. Group3: Delayed liquefaction but with normal viscosity. Group4: Delayed liquefaction and hyperviscosity. Horizontal lines represent median values. P values were determined by the Kruskal-Wallis Test.
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Table 4.4. Correlation between prostatic KLKs
KLK2 KLK3 KLK5 KLK6 KLK7 KLK8 KLK1 KLK1 KLK1 KLK1 0 1 3 4
KLK1 rs .216 .171 .010 .057 -.042 .038 -.077 .137 .106 .466 p .089 .182 .946 .678 .760 .786 .575 .283 .407 <0.00 1
KLK2 rs .361 .648 .408 .208 .318 .569 .484 .251 .495 p .004 <0.00 .002 .128 .019 <0.00 <0.00 .047 <0.00 1 1 1 1
KLK3 rs .142 .072 .068 .132 .173 .204 .153 .145 p .343 .604 .622 .342 .207 .109 .231 .258
KLK5 rs .790 .806 .715 .819 .350 .445 .417 p <0.00 <0.00 <0.00 <0.00 .016 .002 .004 1 1 1 1
KLK6 rs .735 .731 .699 .360 .549 .453 p <0.00 <0.00 <0.00 .007 <0.001 .001 1 1 1
KLK7 rs .533 .695 .152 .457 .290 p <0.00<0.00 .269 <0.001 .032 1 1
KLK8 rs .517 .277 .570 .446 p <0.00.042 <0.001 .001 1
KLK10 rs .297 .493 .525 p .028 <0.001 <0.00 1
KLK11 rs .175 .303 p .171 .016
KLK13 rs .483 p <0.0 01
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4.3.2. Association of KLKs with Liquefaction and Viscosity State
Multivariant logistic regression models were used to examine the potential discriminatory value of the seminal KLKs in determining the state of semen liquefaction
and/or viscosity. We found that a combination of KLKs could improve the classification
capacity of individual KLKs.
In case of semen liquefaction defects, the combination function of 0.41*
LG10(KLK2 ) -0.23* LG10(KLK3) +0.41* LG10(KLK13 ) -0.56* LG10(KLK14) had a
statistically significant discriminatory value (p=0.025) (Fig. 4.2 A)(LG10 = decimal
logarithm).
Similarly, combination of KLK1, 2, 5, 6, 7, 8, 10, 13, and 14, with the function of
1.94*LG10(KLK1) – 1.34* LG10(KLK2 ) +1.64* LG10(KLK5) +0.37* LG10(KLK6 )
+1.92* LG10(KLK7 ) -2.22* LG10(KLK8) -0.35* LG10(KLK10 ) - 0.45*
LG10(KLK13) + 0.43* LG10(KLK14) exhibited a strong discriminatory potential of
semen viscosity status (p<0.001) (Fig. 4.2B).
4.3.3. Association Between Semen Liquefaction State and Variables of Sperm Motility
To determine whether delayed liquefaction has any adverse effects on sperm
movement, several indicators of sperm motility were compared between clinical groups 1
and 3. Number of motile sperms (×106) had a median of 12.7 and 42.3 and mean of 34.1
and 64.2 in samples with delayed and normal liquefaction, respectively. Similarly, the
straight line speed (µm s−1) of sperms had a median of 38 and 47.5 and mean of 40.4 and
48.2 in samples with delayed and normal liquefaction, respectively. We concluded that
both number of motile sperms and straight line speed were significantly decreased (p=
CLINICAL VALUE OF SEMINAL KLKS IN SEMEN ANALYSIS 124
0.017 and 0.026, respectively) in the patient group with delayed liquefaction (Table 4.5).
No appreciable change was detected in either % motility or motile sperm concentration.
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FIGURE 4.2. KLK combination function for the prediction of A). liquefaction and B). viscosity. Multivariate logistic regression models were developed, adjusted only for statistically significant KLK variables. The log likelihood scores were then calculated for each patient. All dependent variables are in their log-transformed state. p values were calculated by the Mann Whitney test.
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Table 4.5. Sperm motility properties in different states of sperm liquefaction Variable Normal Delayed p value* Liquefaction Liquefaction Motile sperm concentration 33.6±9.9 20.9±7.1 N.S Mean±SE 53.6 8.1 Median
% Motility Mean±SE 33.5±2.4 27.5±3.3 N.S Median 30.3 24.7
Number of motile sperms Mean±SE 64.2±13.9 34.1±21.3 0.017 Median 42.3 12.7
Straight line speed Mean±SE 48.2±1.8 40.4±3.27 0.026 Median 47.5 38.0
* Mann Whitney test N.S: Non significant (p>0.05)
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4.3.4. Distribution of Seminal KLKs in Asthenospermic Patients
To examine whether seminal KLKs are differentially expressed in asthnospermic
cases, the concentrations of these KLKs in seminal plasma were compared with cases
with normal percentage (≥ 35%) of sperm motility. Only KLK14 was found to be present
at lower (p=0.022) levels in asthenospermic cases (Fig. 4.3). The mean and median
values of KLK14 concentration (µg/L) in the normal group were 23.3 and 13.5,
respectively. A mean of 9.6 and median of 7.9 were observed in the asthenospermic
group (Table 4.6).
Correlative analysis of KLKs measured in this study and several indicators of ,- ppcorrelation with % motility (Spearman’s rank correlation of -0.283, p= 0.04).
Conversely, KLK14 was found to be positively correlated with percent motility with a
Speraman’s rank correlation of 0.260, p=0.045.
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FIGURE 4.3. Scatter plot of KLK14 levels (µg/L) in the seminal plasma of normal and asthenospermic cases. Individuals with ≤ 35% sperm motility were considered as clinically asthenospermic. p value was calculated by the Kruskal-Wallis test. Horizontal lines represent the median values.
Table 4.6. KLK concentration in Normal and Asthenospermic cases KLKs Normal Asthenospermic p value*
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(ug/L) KLK1 N 24 36 Mean±SE 64.3±9.2 66.6±12.0 N.S Median 62.9 38.5 KLK2 N 24 36 Mean±SE 23660±5270 43510±15890 N.S Median 16650 17080 KLK3 N 24 36 Mean±SE (11±1.2) x106 (5.5±4.0) x106 N.S Median 10x106 11x106
KLK5 N 21 24 Mean±SE 15.0±6.5 17.69±6.0 N.S Median 3.81 7.96 KLK6 N 21 32 Mean±SE 3.34±0.69 4.88±1.43 N.S Median 2.59 1.90 KLK7 N 22 31 Mean±SE 13.3±5.0 12.9±4.1 N.S Median 8.0 5.5 KLK8 N 22 30 Mean±SE 5.69±2.76 8.58±3.9 N.S Median 1.98 2.37 KLK10 N 22 31 Mean±SE 5.16±1.47 6.00±1.74 N.S Median 2.74 3.52 KLK11 N 24 36 Mean±SE 7409±1202 18525±11381 N.S Median 5960 6191 KLK13 N 24 36 Mean±SE 47.3±6.5 44.2±5.8 N.S Median 47.9 40.5 KLK14 N 24 36 Mean±SE 23.3±7.2 9.6±1.5 0.022 Median 13.5 7..9
* Mann-Whitney test N.S: Non-significant (p>0.05)
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Table 4.7. Correlation between prostatic KLKs and indicators of sperm motility Motile sperm % Num. of Straight line concentration (106 cc-1) Motility motile speed (µm s-1) Sperms (106)
KLK1 rs .005 .181 .051 .188 p .972 .167 .727 .205
KLK2 rs -.039 -.026 -.003 -.008 p .766 .846 .983 .959
KLK3 rs -.174 -.108 -.069 .015 p .184 .412 .637 .921
KLK5 rs -.246 -.215 -.177 -.124 p .103 .156 .310 .493
KLK6 rs -.041 -.069 -.040 .035 p .773 .623 .797 .829
KLK7 rs -.180 -.147 -.108 .079 p .198 .294 .490 .626
KLK8 rs -.100 -.112 -.087 .075 p .479 .430 .582 .644
KLK10 rs -.171 -.283 -.156 -.111 p .221 .040 .319 .489
KLK11 rs -.103 -.012 -.058 -.063 p .435 .926 .693 .673
KLK13 rs .074 -.103 .064 -.062 p .574 .434 .663 .680
KLK14 rs .174 .260 .181 .180 p .182 .045 .213 .227
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4.4. DISCUSSION
Semen is primarily the product of secretions from the accessory glands of the male genital tract (199;298). These secretions contain a wide range of substances essential for proper function of the sperm (299). Thus, even though often overlooked, slight changes in the constituents of the glandular portion of semen could have a profound effect on the fertilization potential of the sperm (299;300).
The prostatic secretions are the second largest contributor to the ejaculate, accounting for approximately 10-30% of combined semen volume (199). Secretions of the prostate gland are biochemically highly active, containing key enzymatic components of the ejaculate involved in semen coagulation and liquefaction (199;301). Coagulation is induced upon mixing of different components of the ejaculate, mainly as a mechanical means to trap sperms and render them immobile (265). More recent evidence suggests that various components of the coagulum, including the prostatic zinc ions, may have additional physiological implications in inhibiting sperm motility by directly binding to the surface of the sperm (302). The subsequent process of liquefaction, characterized by the breakdown of the coagulum, is modulated by a number of proteases that are
exclusively expressed in the prostate gland (227;266;303). Given the functional
importance of these prostatic enzymes, reproductive failure could occur as a result of
pathology in the prostate gland and inadequate secretion of these enzymes (210;304).
More recently, several members of the KLK family were suggested to participate
in the process of semen liquefaction, functioning through highly regulated proteolytic
cascades (194;305). According to the comprehensive expression profiling completed
recently, the majority of KLKs (i.e. KLK1-3, 5-8, 10, 11, 13, and 14) are expressed by
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the prostate gland and secreted into seminal plasma in varying amounts (30). Despite
convincing evidence supporting the physiological function of some of the seminal KLKs
in semen liquefaction, clinical data on the pathological role of these KLKs is still lacking.
Similarly, the etiological factors of hyperviscous semen are largely unknown.
Biochemical analysis of semen rheology has previously demonstrated that the observed viscosity is mainly due to highly organized peptide cores complexed with oligosaccharide chains and disulfide bonds (306). Even though the nature of these protein networks still remains to be determined, no measurable difference has been reported in the total protein level of hyperviscous samples, as compared to normal specimens (306). However, treatment of hyperviscous semen with mucolytic enzymes, including trypsin, dithiothreitol, and alpha-amylase resulted in reduced viscosity (306;307). This may suggest that the observed higher consistency of hyperviscous samples could be due to incomplete digestion and persistence of the protein networks. However, to this date, the enzyme(s) that may physiologically be involved in this phenomenon is still unknown. A reduced level of lysozyme was shown to play a possible role in cases of chronic
infections, yet no appreciable difference was observed in normal and hyperviscous semen
with no infection (308), refuting the direct role of lysozyme in this phenomenon.
Given that macroscopically, hyperviscous semen highly resembles abnormal liquefaction and samples can be modified chemically by addition of digestive enzymes, it is conceivable that there may be some overlap between the probable causes of these two abnormalities. Similar to cases of delayed liquefaction, hyperviscosity of semen has been attributed to dysfunctions of male accessory glands (300). For instance, a higher incidence of hyperviscous semen is reportedly associated with hypofunction of the
CLINICAL VALUE OF SEMINAL KLKS IN SEMEN ANALYSIS 133 seminal vesicles (309). Even though still controversial, malfunction of the prostate gland has also been suggested to play a role in the etiology of hyperviscous semen(261;310).
This study was designed to examine the possible role of several members of the KLK family in the pathogenesis of delayed liquefaction and hyperviscosity of semen.
As expected, a number of KLKs, i.e. KLK2, 3, 13, and 14, were found to be aberrantly expressed in individuals with abnormal liquefaction, regardless of their viscosity state. Upon closer examination of the four clinical subgroups, we found a differential dysregulation pattern of KLKs in samples with normal viscosity and delayed liquefaction, as compared to cases of abnormal viscosity and normal liquefaction.
Consistent with the previously published data, KLK3 concentration was found to be reduced in samples with delayed liquefaction but not in those with higher viscosity (291).
However, we observed a significant decline in the expression level of a number of KLKs, i.e. KLK1, 2, 5-8, 10, 13, and 14, in hyperviscous samples with normal liquefaction. This is the first report on abnormal KLK expression in hyperviscous cases, suggesting a common etiologic origin between hyperviscosity and delayed liquefaction.
Furthermore, the aberrant expression patterns of seminal KLKs were found to have diagnostic value in discriminating cases of abnormal liquefaction and viscosity. As mentioned previously, cases of hyperviscous semen may visually be discriminated from delayed liquefaction by looking at the level of conformity of the ejaculate to its environment. However, due to their similar physical appearance and concurrent nature, there is a need for a more accurate diagnostic measure. Here, we have shown the discriminatory value of KLK2, 3, 13, and 14 for cases of delayed liquefaction, while the
CLINICAL VALUE OF SEMINAL KLKS IN SEMEN ANALYSIS 134
combination of KLK1, 2, 5, 6, 7, 8, 10, 13, and 14 appears useful in the diagnosis of
hyperviscous cases.
Finally, given the previous reports of impaired prostate function in cases of poor
sperm motility (300), we next examined various parameters of sperm motility in men
with delayed liquefaction. In order to eliminate the possibility of induced sperm motility
as a result of enzymatic treatment of samples, only untreated samples were considered.
We observed a significant decline in both number of motile sperms and straight line
speed in patients with delayed liquefaction. This is perhaps due to the mechanical constraint imposed by the semen coagulum. Recent evidence suggests that in addition to
its mechanical effect, the semen coagulum physiologically impedes sperm movement
through zinc- mediated accumulation of semenogelin proteins and binding to sperm
surface (302). We did not observe any significant change in motile sperm concentration
or %motility in patients with delayed liquefaction. This could be explained by a lower
number of total sperm in patients with delayed liquefaction, as compared with the normal
control samples (Table 4.1).
Since aberrant proteolytic activity of extracellular proteases has been implicated
in male factor infertility, both in human and a mouse model (311), we next tested whether
seminal KLKs are differentially expressed in cases with impaired sperm motility.
Previously, we reported a lower concentration of KLK14 in asthenospermic individuals,
characterized by ≤35% sperm motility (44). KLK14 seems to be the only member of the
family that is significantly dysregulated in asthenospermic patients. We found a positive
correlation between the expression level of KLK14 and % sperm motility. This is
consistent with previous reports that KLK14 is a highly active trypsin-like protease,
CLINICAL VALUE OF SEMINAL KLKS IN SEMEN ANALYSIS 135 functioning as an activator, at the higher hierarchical level of the proteolytic cascade
(64;67;287). Therefore, we speculate that even though the total protein level of the remaining KLKs is comparable in normal and asthenospermic patients, the level of active enzyme is decreased at least for those KLKs that are activated by KLK14.
In conclusion, KLK concentration in seminal plasma may have clinical utility in the differential diagnosis of delayed liquefaction and hyperviscous cases. In addition, these findings may provide new approaches to identify the pathogenic mechanisms and possible therapies for these two causes of male subfertility. However, given the relatively small size of our cohort, further studies will be necessary to validate our findings.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 136
CHAPTER 5 Identification of a Potential Role of Multiple Members of the Seminal KLK Cascade as Novel Activators of the Latent TGFβ1 Complex in Seminal Plasma
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 137
5.1. INTRODUCTION
Suppression of the female immune response is one of the key processes postulated to be induced by the semen (229). However, given the importance of host protection throughout the female reproductive tract, the immunosuppressive activity of the semen is extremely transient and is tightly regulated, both spatially and temporally. Since sperms are progressively released to the female reproductive tract only after the breakdown of the coagulum, it is conceivable that the two processes of immunosuppression and liquefaction of semen are temporally regulated through common factors.
TGFβ1 is reportedly a major immunosuppressive protein in human seminal plasma (237). Seminal TGFβ1 is primarily secreted to seminal plasma or possibly sequestered to the surface extracellular matrix of the sperm as a latent complex. A number of serine and metalloproteinases have been implicated in various processes leading to activation of the TGFβ family, particularly TGFβ1 in seminal plasma. Given the importance of KLKs in regulation of semen liquefaction and postulated co-regulation of semen liquefaction and immunosuppression, we have investigated possible role of the previously identified seminal KLK cascade, as well as the two highly expressed cervico- vaginal KLKs, in activation of the latent TGFβ1 complex.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 138
5.2. EXPERIMENTAL PROCEDURES
5.2.1. Reagents
Recombinant KLK5, 11, 12, 13, and 14 were produced in house, as described previously (189). Mature KLK1, produced in a baculovirus/insect cell line system, was kindly provided by Dr. M. Blaber (Florida State University, USA). Recombinant KLK2 was a gift from Hybritech Inc. (San Diego, USA). Recombinant human LAP, latent
TGFβ1, anti-human LAP, and anti-human LTBP1 antibody were purchased from R&D systems (Cedarlane Laboratories Limited, Hornby, ON, Canada). Partial recombinant
LTBP1 (403aa-501aa) was purchased from Abnova Corporation (Cedarlane Laboratories
® Limited, Hornby, ON, Canada). TGFβ1 Emax Immunoassay system was purchased from
Promega (Madison, WI, USA).
5.2.2. Enzymatic Activation of TGFβ1
Active recombinant KLKs (final concentration of 6.4 ng/ml for KLK2, 11, 12, and 13, and 14, 3.2 ng/ml for KLK1, and 1.6 ng/ml for KLK5 and 14) were preincubated with 16 ng/ml of the recombinant latent TGFβ1 in 50 µl of the optimal buffer (Table 5.1) at 37 °C with gentle agitation for different time points. Reactions were repeated three times.
KLK14- mediated activation of TGFβ1 was confirmed ex-vivo by preincubating
50 µl of seminal plasma pooled from 10 normal volunteers with 40 and 200 ng/ml active recombinant KLK14 for various amounts of time.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 139
5.2.3. Activation of Latent TGFβ1 by Acid Treatment
Recombinant latent TGFβ1 and seminal plasma were adjusted to approximately pH 2.6, using 1N HCl and incubated at room temperature for 30 minutes. Samples were subsequently neutralized to pH 7.2 by 1N NaOH. Active TGFβ1 was measure in 50µl of
reaction mixtures by ELISA.
5.2.4. TGFβ1 Activity Enzyme-Linked Immunosorbent Assay (ELISA)
® The amount of biologically active TGFβ1 was measured, using the TGFβ1 Emax
Immunoassay system according to the manufacture’s protocol. Briefly, 96 well plates were coated with 10 µl of TGFβ Coat mAb, over night at 4°C. The plate was washed two times in 1×TBST (50mM Tris, 150mM NaCl, 0.01% Tweeen 20, pH 7.5), and blocked for 35 minutes at 37°C, using 270 µl/ well of the 1×TGFβ block buffer. The plate was rewashed two times and loaded with 50 µl of reaction mixtures for 90 minutes at room temperature. After five times wash, 10 µl captured TGFβ1 was bound by a second specific polyclonal antibody. The specifically bound pAb was detected using 100 µl of the TGFβ HRP conjugate, following incubation with 100 µl/well of the chromogenic substrate TMB. Reactions were stopped with 1N HCl and measured at 450 nm with a time-resolved fluorometer (Envision, Perkin- Elmer Corp. Waltham, MA). The background absorbance and residual activity of the recombinant latent TGFβ1 complex were subtracted from raw values of enzyme alone and reaction mixtures, respectively.
The residual activity of KLKs were accounted for by including an additional enzyme alone reaction.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 140
Table 5.1. Description of KLK optimal assay buffers
Buffer composition KLK1 20mM Tris/HCl, 1mM EDTA, 10% DMSO, and 0.1% TritonX-100, pH 9.0
KLK2 10 mM phosphate buffer, pH 7.4
KLK5 100mM phosphate buffer, 0.01% Tween 20, pH 8.0
KLK11 50 mM Tris, 1 M NaCl, 10 mM EDTA, pH 8.5
KLK12 0.1 M Tris, 0.15 M NaCl, 10 mM CaCl2, pH 7.5
KLK13 50 mM Tris, 0.15 M NaCl, pH 8.0
KLK14 100mM phosphate buffer, 0.01% Tween 20, pH 8.0
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 141
5.2.5. Electrophoretic Detection of Mature TGFβ1 Under Native Condition
17.5 ng of active recombinant KLK14 was incubated with 175 ng of recombinant
latent TGFβ1 in 20 µl of optimal buffer at 37 °C with gentle agitation for different time
points. Control reactions of KLK14 and latent TGFβ1 were incubated alone. Reactions
were terminated by freezing in liquid nitrogen and were subsequently resolved by NB-
PAGE, using the NativePAGE Bis-Tris (Invitrogen), with 3-12% gradient
polyacrylamide gels, under native conditions at 150 V for 50 minutes at 4°C. The voltage
was increased to 200V for an additional 5 minutes. Samples were visualized by silver
staining.
5.2.6. In-vitro Cleavage of LAP and LTBP1
Recombinant LAP (300 ng) and LTBP1 (100 ng) were incubated separately with
KLK1, 2, 5, 11, 12, 13, and 14 (gram ratios of 1:2 for KLK2 and 11, 1:5 for KLK1, and
1:10 for KLK5, 12, 13, and 14 ) in a final optimal buffer volume of 20 µl for different
time points at 37 °C (for KLK1, 2, 5, 11, 12, and 13) and room temperature (for KLK14)
with shaking. Control reactions, i.e. KLKs, LAP, and LTBP1 incubated alone, were also
included. Reactions were terminated by freezing in liquid nitrogen and were subsequently
resolved by SDS-PAGE, under reducing conditions at 200 V for 45 min. Samples were
visualized by silver staining.
5.2.7. N-terminal Sequencing of the Newly Generated LAP and LTBP1 Fragments
Recombinant LAP (900 ng) and LTBP1 (300 ng) were incubated separately with
KLK1, 2, 5, and 14 at the gram ratios of 1:2 for KLK2, 1:5 for KLK1, and 1:10 for
KLK5, and 14, in a final optimal buffer volume of 20 µl for 3 hours at 37 °C (for KLK1,
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 142
2, and, 5) and 30 minutes at room temperature (for KLK14). Proteins were electroblotted
to polyvinylidene difluoride (PVD) membrane and stained with Coomassie Blue stain.
Fragments were cut from the membrane and N-terminally sequenced, using the Edman
degradation method.
5.2.8. Western Blotting for Identification of LAP and LTBP1 Fragmentation
KLK-mediated fragmentation of LAP was confirmed in-vitro by incubating
recombinant LAP with active KLK at the abovementioned gram ratios and time points, in
a total volume of 20 µl.To examine fragmentation of LAP and LTBP1 ex-vivo, the pooled
semen coagulum was analyzed by western blot. Recombinant and seminal proteins were resolved by SDS-PAGE, using the NuPAGE Bis-Tris, with 4-12% gradient polyacrylamide gels (Invitrogen) at 200 V for 45 min. Proteins were subsequently
transferred onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 volts
for 1 hour. The membrane was blocked for 1 hour with 5% milk/TBS-Tween [0.1
mol/liter Tris- HCl containing 0.15 mol/liter NaCl and 0.1% Tween 20] at 4°C and
probed using 0.1µg/ ml goat anti-LAP or anti-LTBP1 polyclonal antibody for 1 hour at
room temperature. The membrane was washed three times for 15 minutes with TBS-
Tween and treated with ALP-conjugated monkey anti-goat antibody (diluted 1:5000) for
45 minutes at room temperature. The membrane was re-washed as above, and
fluorescence was detected on X-ray film using a chemiluminescent substrate.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 143
5.3. RESULTS
5.3.1. In-vitro Regulation of TGFβ1 Activity by KLKs
Given that activation of latent TGFβ1 requires cleavage after the trypsin-like cleavage site of Arg 279 (312), we examined whether any of the trypsin-like seminal and/or cervico-vaginal KLKs function as activators of TGFβ1. The ability of KLK1, 2, 5,
11, 12, 13, and 14 to activate recombinant latent TGFβ1was tested. Since mature TGFβ1 exhibits an altered immunoreactivity due to its exposed receptor binding motif, a sandwich capture-ELISA was used to specifically measure latent TGFβ1 activation.
Among tested KLKs, only KLK14 was able to activate latent TGFβ1 in-vitro. At the physiologically relevant 1:10 gram ratio, activation was rapid and transient
(Fig. 5.1), reaching a peak as early as five minutes. The absorbance indicator of active
TGFβ1 increased to a maximum of approximately 50% at 45 pM of enzyme. However, the activation potential of KLK14 was lower than the well-established acid treatment approach. KLK14- mediated activation of latent TGFβ1 was approximately 40% of the activity achieved by acid treatment (data not shown). Following longer incubation times, the amount of biologically active TGFβ1 declined, suggesting a deactivation mechanism that may act as a negative feedback loop. TGFβ1 activation seems to be dependent on
KLK14 concentration, as a lower level of active mature TGFβ1 was detected at a higher concentration of the enzyme (gram ratio of 1:2.5) (Fig. 5.1).
Release of mature TGFβ1 was also confirmed by gel electrophoresis on 3-12%
BN-PAGE, which demonstrated a single band at ~25kD, under native conditions
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 144
(data not shown). Consistent with the above findings, the intensity of the band representing the newly released mature TGFβ1 was reduced following longer incubation with active KLK14.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 145
FIGURE 5.1. In-vitro Activation of the latent TGFβ1. 16 ng/ml of the recombinant latent TGFβ1 was incubated with 1.6 ng/ml of KLK14 and 6.4ng/ml of KLK14 for varying time intervals at 37°C. The activity of TGFβ1 was measured by ELISA and shown as mean ± SD from triplicate assays. The basal activity of TGFβ1 alone for each incubation time has been subtracted. Note the gradual increase in the absorbance followed by reduction of activity. KLK alone bars represent negative controls (no TGFβ1 added).
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 146
5.3.2. KLK14 Mediated Regulation of Endogenous TGFβ1 in Seminal Plasma
In order to confirm KLK14-mediated activation of TGFβ1 complex ex-vivo, seminal plasma samples pooled from healthy volunteers were spiked with 40 and 200 ng/ml active KLK14. The specific activity of TGFβ1 was next examined. Consistent with our in-vitro observations, KLK14 was able to induce TGFβ1 activity almost three folds, two minutes after incubation with seminal plasma (Fig. 5.2). The subsequent decrease in the observed TGFβ1 activity may suggest an inactivation mechanism. A similar pattern was observed when five times more active enzyme was added (Fig. 5.2). However, compared to what was shown in-vitro, the initial level of activation was much lower, possibly due to the presence of excess enzyme and immediate deactivation of the newly released mature TGFβ1.
5.3.3. KLK- Mediated Cleavage of LAP
As mentioned previously, activation of TGFβ1 requires proteolytic cleavage of the propeptide LAP fragment from the latent complex. To examine whether KLK14 activates latent TGFβ1 by fragmentation of the LAP propeptide, recombinant LAP protein was incubated in 10 times excess, with KLK14 for various amounts of time at room temperature. KLK14 was able to almost fully cleave LAP, as quickly as 2 minutes of incubation and completely cleave LAP within the first 15 minutes of incubation
(Fig. 5.3A). Only one of the newly generated fragments was detected by the polyclonal antibody raised against the full-length human LAP protein. This fragment has the N- terminal sequence of VAGESA, cleaved after Arg(58).
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 147
FIGURE 5.2. Activation of endogenous latent TGFβ1 complex in seminal plasma. Seminal plasmas from 10 normal volunteers were pooled and treated with KLK14, in final concentration of 40ng/ml and 200ng/ml. Treated and control samples were incubated at 37°C for varying amounts of time. The activity of TGFβ1 was measured by ELISA and shown as mean ± SD from triplicate assays.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 148 A).
B).
C).
D).
FIGURE 5.3. LAP fragmentation. Recombinant LAP was incubated for varying time intervals with A), KLK14. at 1:10 gram ratio, B). KLK1, at 1:5 gram ratio, C). KLK2, 1:2 gram ratio, and D). KLK5, at 1:10 gram ratio. The mixtures were resolved by SDS- PAGE under reducing conditions and were silver stained (right panel) and immunoblotted using a goat poly anti-human LAP antibody. Molecular mass standards are in KDa. Major KLK-generated fragments are indicated by filled arrowheads. The N- termini of these fragments were consequently sequenced.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 149
In addition, the ability of KLK1, 2, 5, 11, 12, and 13 to cleave LAP propeptide was tested in-vitro. KLK1, 2, and 5 were able to fragment LAP, even though the cleavage efficiency was significantly lower, when compared to KLK14 (Fig. 5.3 B, C, and D).
Interestingly, contrary to KLK14, these KLKs only generated a single new fragment. The fragment produced by KLK5 shared a common N-terminal sequence of VAGESA with that of KLK14. Sequencing results of KLK1 and 2- mediated LAP cleavages were inconclusive. This was more likely due to the low cleavage efficiency by these enzymes and lack of sufficient amount of newly generated fragments required for sequencing. No
cleavage was observed when LAP was incubated with KLK11, 12, or 13, at various
concentrations and time points (data not shown).
5.3.4. Fragmentation of Endogenous LAP in Seminal Plasma
Given that several members of the KLK family were able to proteolytically cleave
the propeptide (LAP) motif of the recombinant latent TGFβ1 complex, we next examined
LAP fragmentation pattern ex-vivo in seminal plasma. There were two major LAP-related
fragments identified by western blotting, using goat anti-LAP polyclonal antibody. In
addition to the expected 35kDa band that supposedly represents the full-length
propeptide, only one band with molecular weight of approximately 28kD was identified.
Interestingly, comparing with our previous in-vitro results, only KLK14 was able to
generate a fragment with the molecular mass of this band (Fig. 5.4).
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 150
FIGURE 5.4. LAP fragments in seminal plasma. Several amounts of seminal plasmas from 10 normal volunteers were pooled. Various amounts of the pooled sample were run under reducing conditions and immunoblotted, using a goat polyclonal human LAP antibody. SP, seminal plasma. Note the similar fragmentation pattern observed in-vitro (Fig. 5.3A) and ex-vivo.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 151
5.3.5. KLK- Mediated Cleavage of LTBP1
Finally, KLKs may be involved in solublizing TGFβ1 complex by releasing it from its binding protein, LTBP. This possibility was tested by incubating recombinant
LTBP1 with the abovementioned KLKs in various molar ratios and time points. Similar to LAP fragmentation, KLK1, 2, 5, and 14 cleaved LTBP1, while no cleavage was observed upon incubation with KLK11, 12, and 13 in various tested molar ratios and time points (Fig. 5.5). The cleavage efficiency of the effector KLKs varied significantly, with
KLK14 as the most and KLK1 as the least time efficient enzyme. In addition, KLK14 seemed to generate a larger number of new fragments. This was followed by rapid peptidic fragmentations of the newly formed fragments, as their respective bands disappeared in longer incubation points. Unfortunately, due to this technical difficulty, we were unable to sequence the newly formed fragments. As for other KLKs, the N- terminal protein sequencing failed to detect the cleavage sites possibly due to their blocked N-terminus amino acids. It should be noted that since the recombinant LTBP1 contains only a partial sequence (403aa-501aa), additional processing of the full length protein is highly probable.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 152
FIGURE 5.5. LTBP1 fragmentation.100ng of recombinant LTBI was incubated for varying time intervals with A). KLK1, 1:5 gram ratio, B). KLK2, 1:2 gram ratio, C). KLK5, 1:10 gram ratio and D). KLK14, 1:10 gram ratio. The mixtures were resolved by SDS-PAGE under reducing conditions and were silver stained (right panel). Molecular mass standards are in KDa. Major KLK-generated fragments are indicated by filled arrowheads. These fragments (except those indicated in part D) were N-terminally sequenced.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 153
Based on the information provided above, a putative model was developed for
KLK activation of seminal TGFβ1 complex (Fig. 5.6). It is probable that other KLKs and/or other classes of enzymes may also participate in this pathway.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 154
FIGURE 5.6. Schematic presentation of the proposed functions of multiple KLKs in activation of TGFβ1 complex. Active KLKs are involved in activation of the latent TGFβ1 through cleavage of LAP or its binding protein LTBP1. KLK1, 2, 5, and 14 are suggested to cleave LAP at a single site, causing a more open conformation. Further cleavage of the LAP propeptide by KLK14 and a complete dissociation of the LAP fragment would eventually lead to release of mature TGFβ1.
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 155
5.4. DISCUSSION
TGFβs are involved in a variety of cellular processes through activation of their latent forms and interaction of the mature homodimers with their specific receptors. Since
TGFβ receptors are expressed ubiquitously (313), the activation step is regarded as the main control point of TGFβ function. Even though activation can be achieved in-vitro by
acid treatment (314) or treatment with several other chaotropic agents (315), mechanisms
involved in the physiological activation of TGFβ is not fully understood. In particular in seminal plasma, only a small portion of TGFβ1 is reportedly active prior to insemination
(237). In mice, only 30% of TGFβ is active at its site of expression in seminal vesicle
(316). However, more than 70% of TGFβ was shown to be active in uterine fluids after insemination (316). This suggests that activation most likely occur upon ejaculation or after deposition in the female reproductive tract (237). Given the importance of immune response in the female tract and considering the main function of TGFβ in seminal plasma in attenuating host immune response, it is highly likely that activation is triggered only when sperms are released from semen coagula at the time of liquefaction.
To date, several activation mechanisms have been proposed through proteolytic cleavage of the propeptide region (LAP) or the binding protein (LTBP) of TGFβ complexes (237). Here, for the first time, we propose a potential role of several seminal
KLKs in activation of TGFβ1 in seminal plasma. Since TGFβ1 is the predominant isoform recognized in seminal plasma (241), we chose to focus only on this isoform of the family.
Surprisingly, among all seminal and cervico-vaginal KLKs tested, only those
KLKs that belong to the previously identified seminal liquefaction cascade (287) seemed
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 156
to be involved in the activation of latent TGFβ1. Consistent with our findings that
KLK14 is the key player in semen liquefaction (317), KLK14 seems to be the only
seminal KLK directly involved in TGFβ1 activation. This is in accordance with previous reports that KLK2 and 3 were not able to directly activate latent TGFβ1 complex purified
from seminal plasma (254). The observed KLK14- mediated activation seems to occur
through degradation of the TGFβ1 amino-terminal propeptide LAP fragment. The result
of our N-terminal sequencing suggests that the fragment, detected both in-vitro and ex-
vivo in seminal plasma, was generated by cleavage after Arg (58). This is expected, given
that KLK14 exhibits a strong preference for arginine and tyrosine at the P1 position
(189).
KLK14 may also be involved indirectly by increasing the bioavailability of
soluble latent TGFβ1 through cleavage of its binding protein, LTBP1 and release of the
membrane-bound complex. ECM- bound TGFβ1 complexes have been reported as a
primary storage and targeting site in the female reproductive tract, including in the
vagina, cervix, and endometrial wall (318-321). The significance of membrane-bound
TGFβ1 in the male reproductive system is less understood, as the majority of seminal
TGFβ1 is reportedly in the soluble form (322). However, according to UniProtKB/Swiss-
Prot and GenAtlas, LTBP1 is synthesized at a high level in the male reproductive system,
mainly by the prostate. Furthermore, immunohistochemical studies of human
spermatozoa indicates TGFβ1 localization at the postacrosomal region of the head, the
neck, and the middle segment of the tail (322). Whether this binding is through LTBP
remains to be investigated. However, the majority of TGFβ1 complexes purified from
seminal plasma reportedly ranged from 100-440kDa (254). This may suggest that the
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 157
short latent complex, consisting of the mature TGFβ1 and LAP propeptide homodimers, are bound to additional components. Given that the larger isoforms of TGFβ1 complexes,
which contain LTBPs, have a molecular weight of approximately 300kDa (250), it is
possible that the larger detected seminal TGFβ1 is associated with LTBP. In fact, we
observed several fragments in seminal plasma immunoblotted against LTBP1 antibody
(data not shown). Whether these are true cleavage products of LTBP1 shed into seminal
plasma, or nonspecific fragments detected by the polyclonal antibody needs to be further
examined.
LTBP1 is associated non-covalently to the N-terminal LAP propeptide via a
disulfide bound to its third 8-cysteine domain (323). In addition, even though still not
validated, based on similarity prediction, LTBP1 may covalently binds to the latent
TGFβ1 complex through its third TB (TGF binding) domain. Proteolytic cleavage of
LTBP1 and the subsequent secretion of the TGFβ1 complex has been reported in various
physiological systems, in particular in osteoclasts, through a number of enzymes including plamsin, elastase, MMP-2 and 9 and TSP-1 (250;324;325).
Analogous to KLK14, KLK1, 2, and 5 may function in LTBP1 cleavage.
Unfortunately our attempt to sequence KLK cleavage sites failed. We suspect that this may be due N-terminal blockage of the fragments, as the N-terminus of LTBP1 has been reported to be blocked, according to the UniProt protein database. As mentioned previously, whether LTBP1 plays a role in semen is still questionable. However, given the importance of LTBP in the female reproductive tract, we speculate that the proposed processing of LTBP1 has functional significance in signalling in the female reproductive system. The observation that none of the two highly expressed KLKs in the cervico-
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 158
vaginal fluids, KLK12 and 13 (326), were able to cleave LTBP1 further supports this hypothesis.
Furthermore we propose an alternative function for KLK1, 2, and 5 as accessory factors involved in conformational changes, necessary for making the propeptide LAP motif accessible for further proteolytical processing. A similar function has been suggested for plasmin, where at lower concentrations it only cleaves LAP at the dibasic cleavage site (327). Even though this process was reported to be necessary, it was not sufficient for activation, as the fragmented LAP remained noncovalently associated to the mature TGFβ1 (327). Given that KLK1, 2, and 5 generated a single fragment upon cleavage of LAP, yet were unable to activate the recombinant latent TGFβ1 complex, we
speculate that they may function in a similar fashion as low plasmin concentration.
According to the plasmin activation model (327), complete release of mature TGFβ1 achieves only after further fragmentation of the LAP propeptide, at the higher concentration of the enzyme. It is conceivable that KLK1, 2, and 5 cleave LAP at a single peptide bond to conformationals open up the propeptide for further cleavage by KLK14 and other proteases.
In addition, the single fragment generated by KLK5 and the most prominent
KLK14-produced fragment have different molecular weight but share a common N- terminal sequence. Therefore, KLK14 may also be involved in the initial nicking process.
Further fragmentation by KLK14 in this case is more likely occur at the C-terminus of the nicked LAP propeptide.
In summary, the present study provides strong evidence for a novel function of multiple members of the KLK family in the activation of latent TGFβ1 in seminal
NOVEL ROLE OF SEMINAL KLKS AS ACTIVATORS OF TGFβ1 159
plasma. Given the importance of TGFβ1 and its associated components, including its binding protein LTBP1, as key immune regulating agents in female reproductive physiology, it is plausible that seminal KLKs play a role as novel male factor signalling factors in the female reproductive tract to promote sperm survival.
SUMMARY AND FUTURE DIRECTIONS 160
CHAPTER 6 Summary and Future Directions
SUMMARY AND FUTURE DIRECTIONS 161
6.1. SUMMARY
This thesis has successfully established novel proteolytic activation cascades within several members of the KLK family, in which KLK14 acts as a novel activator.
The proteolytic events elicited by members of one of these cascades have further been expanded in seminal plasma. Below is a summary of the key findings of this study.
6.1.1. Key Findings
KLK activation cascade models downstream of KLK14
The screening approach of a library of the activation motifs of KLKs was implemented to identify potential KLK activation cascades mediated by KLK14. Two putative models with potential function in the skin and seminal plasma homeostasis were proposed. The KLK components of these cascades are postulated to be KLK1, 2, 3, 5, 11, and 14 in seminal plasma and KLK1, 5, 7, 11, and 14 in the skin. This is based on the following findings:
a. Heptapeptides encompassing activation motifs of KLK2, 3, 5, and 11 were
cleaved with a high (≥ 85%) cleavage efficiency.
b. Pro-KLK11, -KLK3, and –KLK1 were rapidly activated in a concentration-
dependent manner.
c. ProKLK3 regulation was bidirectional, as activation was followed by
inactivation via internal cleavage of active KLK3.
SUMMARY AND FUTURE DIRECTIONS 162
Biological role of the proposed seminal KLK cascade in semen liquefaction
Given the involvement of several members of the proposed model of the seminal
KLK cascade in semen liquefaction, possible cascade-mediated role of KLK14 in this process was examined. Our results show that KLK14 exerts a significant and dose- dependent effect in the process of semen liquefaction, as we found:
a. KLK14 expression was significantly lower (p= 0.0252) in individuals with
clinically delayed liquefaction. Concordantly, KLK14 expression was
significantly (p= 0.0478) lower in asthenospermic cases.
b. Specific inhibition of KLK14 activity by the synthetic inhibitor ACTG9 resulted
in a significant delay in semen liquefaction, a drop in the “early” (30 minutes
post-ejaculation) “chymotrypsin-like” and KLK1 activity and an increase in the
“late” (90 minutes post- ejaculation) “chymotrypsin-like”activity. Conversely,
addition of recombinant active KLK14 facilitated the liquefaction process,
augmented the “early” “chymotrypsin-like” activity, and lowered “late”
“chymotrypsin-like” activity.
c. Given that the observed “chymotrypsin-like” activity was almost completely
attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally.
d. A higher level of KLK3 fragmentation was observed in KLK14- induced
coagula, suggesting an inactivation mechanism via internal cleavage.
SUMMARY AND FUTURE DIRECTIONS 163
e. Semenogelins I and II were directly cleaved by KLK14.
f. Semenogelins were also able to reverse KLK14 inhibition by Zn2+, providing a
novel regulatory mechanism for KLK14 activity.
Association between seminal KLKs, abnormal liquefaction, and other macroscopic indicators of semen analysis
Clinical value of seminal KLKs in differential diagnosis and etiology of abnormal liquefaction and hyperviscosity was next examined. Given their differential expression pattern, KLKs may aid in more accurate evaluation of semen quality, based on the following observations:
a. Combination of KLK2, 3, 13, and 14 and KLK1, 2, 5, 6, 7, 8, 10, 13, and 14
showed very strong discriminatory potential for semen liquefaction and viscosity,
respectively.
b. Liquefaction state was significantly associated with two main parameters of
sperm motility, i.e. number of motile sperms and straight line speed.
c. Among all the KLK tested, only KLK14 was found to be differentially
expressed in asthenospermic cases.
SUMMARY AND FUTURE DIRECTIONS 164
Novel biological role of the proposed seminal KLK cascade in activation of latent TGFβ1 in seminal plasma
Given the functional significance of the seminal KLK cascade in semen liquefaction and the proposed co-temporal regulation of immune-suppression and liquefaction in semen, the ability of several KLKs to activate the key immune deviating agent of seminal plasma, TGFβ1, was investigated. Multiple members of the seminal
KLK cascade were found to be directly or indirectly involved in activation of latent
TGFβ1 in seminal plasma, since:
a. Latent TGFβ1 was rapidly activated by KLK14 in a concentration- dependent
manner, both in-vitro and ex-vivo in seminal plasma.
b. The LAP propeptide motif of the small latent TGFβ1 complex was cleaved by
KLK14 into small peptide fragments, providing a possible mechanism for KLK14
activity.
c. KLK14 may also play a role in release of latent TGFβ1 via fragmentation of the
LTBP1 component of the large, membrane-bound complex.
d. Additional members of the cascade, i.e. KLK1, 2, and 5, may indirectly
involved in TGFβ1 activation by proteolytically inducing conformational changes
in LAP that will aid in its subsequent processing or through LTBP1 cleavage.
SUMMARY AND FUTURE DIRECTIONS 165
6.1.2. Conclusion
Reproductive tissues are very unique in that they represent the only physiologic site where allogenic interactions can occur naturally. This is achieved by the ability of
semen to avert immune-mediated damages of the female reproductive tract to ensure
sperm survival. At the time of insemination, the immune-regulatory effect of semen is
more likely induced simultaneously with release of progressive sperms following
liquefaction of semen coagulum to allow successful fertilization.
This thesis describes a novel proteolytic activation cascade within multiple
members of the KLK family that may concurrently regulate the two key processes of
semen liquefaction and immune-suppression through activation of TGFβ1 (Fig. 6.1).
SUMMARY AND FUTURE DIRECTIONS 166
continued…
SUMMARY AND FUTURE DIRECTIONS 167
FIGURE 6.1. Schematic presentation of the proposed cascade- mediated functions of seminal KLKs. The seminal KLK cascade is consisted of a number of KLKs that function at different levels of the cascade. The cascade contain several regulatory mechanisms through endogenous inhibitors (Inh), Zn2+, internal cleavages, and positive/negative feedback loops. Active KLKs are postulated to concurrently engage in the two key processes of liquefaction and immunosuppression in the seminal plasma. Semen liquefaction occurs as a result of proteolytic processing of 1). SgI/II by KLK3, 5, 11, and 14. 2). FN by KLK5 and 14. The immunosuppressive function of seminal KLKs is mediated through the proteolytic cleavage of 1). LTBP1 by KLK1, 2, 5, and 14. 2’). LAP by KLK14 (complete fragmentation) and/or KLK1, 2, 5 (partial digest), and 3’). release of the mature TGFβ1 dimer by KLK14.
SUMMARY AND FUTURE DIRECTIONS 168
6.2. FUTURE DIRECTIONS
This thesis is hoped to provide a framework for future research to further explore the complexity of regulatory events essential for activation and biological function of the
KLK family. Particularly, with the information provided here, future research can be directed to expand the current cascade models and to examine the (patho)physiology and clinical utility of these cascades in various biological systems.
In order to identify a complete theoretical activation cascade model, additional screenings, using the remaining KLKs, are suggested. This could be accomplished systematically by screening of the previously mentioned library with KLKs that may function downstream of KLK14 in the current cascade model. Newly identified activator components could be used as reference points for further screening of their downstream targets. Such a step-wise screening approach would allow for a complete identification of putative components of the model to the final execution level. Following validation of positive hits of the screening, new components could be incorporated to the current cascade model.
To further delineate (patho) physiological functions of the KLK cascade in semen liquefaction, a similar approach as described in Chapter 3 could be applied to specifically inhibit the enzymatic activity of other members of the cascade, in particular those that may function at the higher level of initiation and progression, e.g. KLK2 and 5.
Reciprocally, enzymatic activity of KLK14 and other potential KLK activators could be induced in semen samples that show no or low level of endogenous expression of these
KLKs. As described previously, the effect of these treatments on semen liquefaction rate and enzymatic activity of their downstream targets could next be examined.
SUMMARY AND FUTURE DIRECTIONS 169
With respect to the clinical utility of KLKs in diagnosis of male factor subfertility, a larger sample size is required to validate findings presented in this work. Additional correlative studies to better elucidate the pathogenesis of delayed liquefaction and hyperviscosity is suggested. For instance, possible correlation between KLK expression level and markers of various components of male reproductive tract needs to be investigated, in order to address the question of etiology of these conditions. Additional information pertaining to clinical findings at the time of examination, as well as microscopic, and biochemical analysis of semen needs to be collected and included in future studies.
Given the promising data on potential role of the seminal KLK cascade in the activation of latent TGFβ1, future studies are recommended to validate and expand the current knowledge. For those KLKs that sequencing failed, mapping of KLK-mediated cleavages of LAP and LTBP1 could be accomplished by attempting to unblock the newly formed fragments, using various approaches described elsewhere (328-330), or by using more sensitive sequencing approaches, such as Edman sequencing coupled with accelerator mass spectrometry or dansyl-Edman sequencing methods (331;332). To further explore the possibility of seminal KLKs acting on TGFβ1 complexes of the female reproductive tract, particularly through LTBP1 cleavage, a cell-culture based approach is required. The vaginal epithelial cell line VK2/E6E7 expressing LTBP1- bound TGFβ1 complex is suggested.
The proposed function of KLK14 as an activator of TGFβ1 in the reproductive system could further be investigated in-vivo. Mouse models with RNAi- mediated knockdown of KLK14 or prostate-specific KLK14 knockout mouse models could be
SUMMARY AND FUTURE DIRECTIONS 170
developed. TGFβ1 expression could be induced in these animal models and their wild
type counterparts, using the recombinant viral gene transfer approach. Activation of
latent TGFβ1 is expected to be higher in the wild type population. However, given the
functional importance of TGFβ1 in reproductive system, loss of KLK14 activity is more
likely to be compensated by additional complementary activator components. Therefore,
KLK14 null animals are expected to exhibit a much milder phenotype than TGFβ1 null
models. Unfortunately, since TGFβ1 null mice do not survive to reproductive age, a direct comparison of the phenotype of KLK14 and TGFβ1 null animals is not possible.
Finally, emerging evidence indicates an alternative αVβ6 integrin-dependent
mechanism of TGFβ activation in the lung through PAR1 (333). Interestingly, the uterine
luminal epithelium expresses the highest level of αVβ6 integrin, unparallel to any other
epithelia in primates (334). As mentioned previously, several members of the KLK
family, including KLK14, has been suggested to activate PAR1 and 2, with preference to
PAR2 (335). PAR-mediated involvement of KLK14 and other seminal and/or cervico-
vaginal KLKs in TGFβ1 activation could further be investigated.
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APPENDIX 192
CHAPTER 8 Appendix
APPENDIX 193
FIGURE A1. Schematic presentation of KLK locus and their potential utility as
cancer biomarkers. Listed biomarker applications have been reported based on
differential expressions of respective genes/proteins. Note the concurrent dysregulation of
adjacent KLK genes in several cancer types, suggesting transcriptional regulatory
mechanisms of groups of genes through common promoter regions.
APPENDIX 194
Table A.1. Multiparametric models of KLK and other biomarkers in human cancers
Cancer Tissue/fluid Model KLK and other biomarker panels Clinical relevance AUC* Ref. Solid tumour LR† CA125, B7-H4, KLK7,10,11, 13 Distinguishing primary 0.97 (106) tumours from normal
Ascites and pleural LR KLK5-8, 10, 11, 13, 14 Distinguishing primary 0.99 (105) effusion tumours from normal Ovarian (105) Ascites and pleural LR KLK5-8, 10,11, 13, 14 Distinguishing primary 0.96 effusion tumours from other cancers
Solid tumour LR CA125, B7-H4, KLK4, 5, 7, 8, 11 Distinguishing primary 0.92 (106) tumours from benign
Solid tumour LR CA125, KLK8, 10, 13 Distinguishing primary 0.84 (106) tumours from non- ovarian metastatic tumours
(106) Solid tumour LR KLK6, 8, 11, 13 One-year free survival 0.76 progression predictor
Solid tumour LR B7-H4, KLK6, 7, 11, 14 Five-year free survival 0.76 (106) progression predictor
Solid tumour LR KLK6, 8, 13 Response to chemo- 0.75 (106) therapy
APPENDIX 195
Table A1 (continued) Cancer Tissue/fluid Model KLK and other biomarker panels Clinical relevance AUC* Ref Lung Serum LR KLK4, 8, 10, 11, 12, 13, 14 Distinguishing cancer 0.90 (107) cases from normal Serum with PSA LR KLK2/fPSA and fPSA/tPSA Distinguishing cancer 0.72 (102;1 ranging from 2- cases from BPH 04) 10ng/ml
‡ 0.72 (tPSA, Serum with PSA ANN tPSA,f/tPSA, KLK2, KLK2/fPSA, Distinguishing cancer 1-4ng/ml) (102;1 ranging from 1- KLK2/(fPSA/tPSA) cases from BPH 0.74 (tPSA, 04) 20ng/ml 2-4ng/ml)
(tPSA, 0.78 Prostate 4-10ng/ml) (103) 0.83 (tPSA, Serum with PSA LR tPSA, %fPSA, MIF, MIC-1, KLK11, age, Distinguishing cancer 2-20ng/ml) ranging from 0.5- prostate volume (if available) cases from BPH 20ng/ml (103) 0.85 (tPSA, 0.5-20ng/ml) Serum with PSA ANN tPSA, %fPSA, MIF, MIC-1, KLK11, age, Distinguishing cancer 0.83 (tPSA, ranging from 0.5- prostate volume (if available) cases from BPH 2-10ng/ml) 20ng/ml 0.87 (tPSA, 0.5- 20ng/ml)** 0.83 (tPSA, 2-10ng/ml)**
0.86(tPSA, 0.5-20ng/ml) 0.84(tPSA, 2- 10ng/ml) 0.91tPSA, 0.5- 20ng/ml)** 0.88(tPSA, 2- 10ng/ml)**
* Uncorrected area under curve (AUC), † LR: Logistic regression, ‡ ANN: Artificial neural network, ** Groups with prostate volume available