DOCSLIB.ORG

Down Regulation of KLK7 Expression in Breast Tissues and Identification of a Novel Spliced KLK7 Mrna

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Down Regulation of KLK7 Expression in Breast Tissues and Identification of a Novel Spliced KLK7 Mrna Ejaz et al. Applied Cancer Research (2017) 37:35 Applied Cancer Research DOI 10.1186/s41241-017-0042-8 RESEARCH ARTICLE Open Access Down regulation of KLK7 expression in breast tissues and identification of a novel spliced KLK7 mRNA Samina Ejaz1, Faiz-ul-Hassan Nasim2*, Muhammad Ashraf2 and Gulzar Ahmad3 Abstract Background: Alternative splicing commonly occurs in cancer cells and many cancer specific splice variants have been reported as potential candidate biomarkers of the disease. We have studied human tissue Kallikrein 7 (KLK7) mRNA expression profile in breast cancer patients of our region. KLK7 is member of a multi-gene family consisting of 15 members (KLK1-KLK15). Methods: We optimized touch down nested PCR method for the amplification of KLK7 isoforms/variants. Various bioinformatics tools were used for sequence analysis, identification of splicing pattern and prediction of encoded proteins. Results: We observed an unusual splicing event consisting of exon 3 (E3) truncation at 3′ end (by 124 nucleotides), exon 4 (E4) exclusion and exon 5 (E5) truncation at 5′ end (by 33 nucleotide) in 2 normal breast tissues, one obtained from invasive ductal carcinoma grade II patient and other collected from mammary dysplasia patient. Moreover, 3 other KLK7 mRNAs (KF963190, KF963191, and KF963193) expressed in breast cancer were noticed to exhibit single nucleotide polymorphism (SNPs). Bioinformatic analysis revealed that the alternatively spliced mRNA (KF963192) will potentially encode a truncated and non-functional protein. Similarly although encoded proteins have considerable homology with normal hK7 protein, SNPs seem to cause great variations in pIs, structures and molecular weights of encoded proteins. Conclusions: There is need to further explore the impact of the unique splicing event, SNPs and characterize these population specific mutations and their possible role in the pathogenesis of breast cancer. Keywords: Alternative splicing, Biomarker, Breast cancer, KLK7, Splice variants Background Human tissue Kallikreins (KLKs) is a multigene family Alternative splicing is a way to produce functionally consisting of 15 members which encode serine prote- diverse proteins from a single gene [1, 2]. It occurs in a ases. All KLKs are known to produce alternative tran- variety of biological processes including developmental, scripts through the use of alternative, transcriptional physiological and pathogenic pathways [3]. According to start site, polyadenylation and splicing. Browsing of the one estimate at least 15% of the genetic diseases are the Genbank database revealed that KLKs have 82 reported outcome of defective pre–mRNA splicing and alternative transcripts and 49 (~ 27% of 82) are the outcome of splicing is a common phenomenon occurring in cancer alternative splicing. Some of the splice variants are cells. Various cancer–specific splice variants have been tissue, developmental stage, stimulus or disease specific. proposed as potential biomarkers of cancer [4–6]. Few KLK splice variants help in cancer diagnosis and prognosis [7–9] and can be used as potential biomarkers of cancer [10, 11]. Future work in this area will be particularly helpful to find more useful diagnostic and * Correspondence: [email protected] 2Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, prognostic biomarkers [12]. Pakistan Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ejaz et al. Applied Cancer Research (2017) 37:35 Page 2 of 13 KLK7 is a member of KLKs family and is known to Mean age of patients was 47.32 ± 12.16 years (range encode human stratum corneum chymotryptic enzyme 24–75 years) and majority of patients were suffering (hSCCE of PRSS6) and expressed in a wide range of from infiltrating ductal carcinoma (53.20%). Total 118 tissues [13–15]. According to GenBank, a total of 11 dis- breast tissue samples, 63 diseased and 55 normal tinct transcripts have been reported for KLK7 (Table 1 samples were subjected to analysis. Normal tissues were and Fig. 1) and only three can be categorized as alterna- carefully removed by a surgeon from regions away from tively spliced transcripts/isoforms. KLK7 mRNA expres- area of cancerization. However, in case of 8 patients due sion level has been quantified by RT-PCR in breast to the involvement of whole breast it was not possible to cancer tissues and reported to be a better prognostic obtain normal tissue. Following the instructions of man- marker for the unfavorable prognosis of breast carcin- ufacturers tissue samples submerged in RNAlaterR were oma. KLK7 mRNA was significantly lowered in either transported from hospital to laboratory, incubated over- stage I or stage II breast cancer patients and higher level night at 2-8 °C followed by the removal of tissues from of KLK7 mRNA was found to be associated with better RNAlaterR and storage at -70 °C. prognosis [16, 17]. Breast cancer is the second leading cause of death Extraction and quantification of RNA worldwide [18]. In Asia Pakistan has the highest preva- Tissue samples were processed for the extraction of total lence of breast cancer. On the average about 40,000 RNA using Norgen Biotek corporation ‘total RNA purifi- women die per year due to this disease [19, 20] and one cation kit’ (Catalogue No. 17200). RNA was quantified out of every nine women in Pakistan is at the risk of de- by measuring absorbance (A) at 260 nm and purity of veloping breast cancer [21]. Contrary to western females, RNA was checked by measuring the ratio of A260/280. Pakistani females suffer from the breast cancer at an RNA samples having A260/280 ratio between 1.8–2.1 early age having a higher risk of metastatic cancers and were further subjected to PCR amplifications. are presented with large lesions [22]. Considering the importance of the issue and associ- cDNA synthesis ation of KLK7 mRNAs with breast cancer we hypothe- cDNA was synthesized by reverse transcription using M- sized that based on genetic differences there can be MLV (Moloney Murine Leukemia Virus) reverse tran- certain new splice variants expressed in breast cancer scriptase (Invitrogen, catalogue no. 28025–013). Reverse (BC) patients of our region. The project was therefore transcription was carried out in a reaction mixture of initiated to evaluate the potential of KLK7 isoforms/vari- 20 μl. Initially RNA solution containing 3 μg RNA, ants as biomarkers of breast cancer in this region. This 1.5 μlof20μM poly dT primer (1.5 μM), 1 μlof10mM study is the first to report KLK7 isoforms/variants in dNTPs mix (0.5 mM) were mixed in 0.2 ml PCR tube patients of southern Punjab, Pakistan. placed on ice. Total volume was adjusted to 12.5 μl with nucleases free H2O, if required. Mixture was heated at Methods 65 °C for 5 min and quickly chilled on ice. After cooling The study was approved by appropriate institutional the contents of tube were collected by brief centrifuga- technical committees including the departmental Ethics tion. It was followed by addition of 6.5 μl RT mix and Research Committee and by the Advanced Studies containing 4 μl of 5× first strand buffer (supplied by and Research Board (ASRB) of the Islamia University of Invitrogen with M-MLV reverse transcriptase), 2 μl 0.1 Bahawalpur, Pakistan. Written Consent to participate in MDTT (10 mM) and 0.5 μl nucleases free H2O. the study was taken on a prescribed form from all Contents of tubes were gently mixed by pipetting and patients included in the study. The experimental work incubated at 37 °C for 2 min. Finally 1 μl (200 units) M- involving humans was in complete compliance with the MLV reverse transcriptase (M-MLV RT) was added to Helsinki Declaration. the reaction mixture, contents were gently mixed by Table 1 Already known KLK7 alternative transcripts Sr. no. Constitutively spliced mRNAs Constitutively spliced Alternatively spliced mRNAs Alternatively spliced mRNAs having SNP mRNAs having SNP 1 AY601109.1 NM_005046.2 AY646152.1 AF411215.1 2 NM_139277.1 BC032005.1 AK295313.1 ———— 3 ———— AF411214.1 ———— ———— 4 ———— AK289660.1 ———— ———— 5 ———— L33404.1 ———— ———— 6 ———— DQ018784.1 ———— ———— Ejaz et al. Applied Cancer Research (2017) 37:35 Page 3 of 13 Fig. 1 Alternative transcripts of KLK7. Sequences of transcripts were retrieved from GenBank, aligned again KLK7 gene sequence to deduce KLK7 mRNA’s architecture. All transcripts are drawn in the 5′ -3′ direction pipetting up and down and incubated at 37 °C for In this technique annealing temperature is initially kept 50 min. After 50 min of incubation at 37 °C M-MLV RT above the melting temperature of primers being used for was inactivated by heating at 70 °C for 15 min and the amplification and then over the course of successive cycles reaction mixture was stored at either 4 °C (for short transitions are progressively made to a lower annealing term storage) or -70 °C for long term storage. temperature. TD PCR is better employed to amplify tran- scripts which are difficult to amplify. Moreover, TD-PCR is Designing/selection of primers for PCR amplification helpful for rapid optimization of PCR conditions avoiding Primers for PCR amplification of KLK7 and β-actin lengthy experimentation for optimization [23]. (used as internal control) cDNAs were selected from a In nested PCR 2 sets of primers are used and PCR list of possible suitable primers predicted by the primer amplification is done in two successive runs of PCR.
Load more

Recommended publications
  • Identification of New Substrates and Physiological Relevance
    Université de Montréal The Multifaceted Proprotein Convertases PC7 and Furin: Identification of New Substrates and Physiological Relevance Par Stéphanie Duval Biologie Moléculaire, Faculté de médecine Thèse présentée en vue de l’obtention du grade de Philosophiae doctor (Ph.D) en Biologie moléculaire, option médecine cellulaire et moléculaire Avril 2020 © Stéphanie Duval, 2020 Résumé Les proprotéines convertases (PCs) sont responsables de la maturation de plusieurs protéines précurseurs et sont impliquées dans divers processus biologiques importants. Durant les 30 dernières années, plusieurs études sur les PCs se sont traduites en succès cliniques, toutefois les fonctions spécifiques de PC7 demeurent obscures. Afin de comprendre PC7 et d’identifier de nouveaux substrats, nous avons généré une analyse protéomique des protéines sécrétées dans les cellules HuH7. Cette analyse nous a permis d’identifier deux protéines transmembranaires de fonctions inconnues: CASC4 et GPP130/GOLIM4. Au cours de cette thèse, nous nous sommes aussi intéressé au rôle de PC7 dans les troubles comportementaux, grâce à un substrat connu, BDNF. Dans le chapitre premier, je présenterai une revue de la littérature portant entre autres sur les PCs. Dans le chapitre II, l’étude de CASC4 nous a permis de démontrer que cette protéine est clivée au site KR66↓NS par PC7 et Furin dans des compartiments cellulaires acides. Comme CASC4 a été rapporté dans des études de cancer du sein, nous avons généré des cellules MDA- MB-231 exprimant CASC4 de type sauvage et avons démontré une diminution significative de la migration et de l’invasion cellulaire. Ce phénotype est causé notamment par une augmentation du nombre de complexes d’adhésion focale et peut être contrecarré par la surexpression d’une protéine CASC4 mutante ayant un site de clivage optimale par PC7/Furin ou encore en exprimant une protéine contenant uniquement le domaine clivé N-terminal.
    [Show full text]
  • An Overview of the Kallikrein Gene Families in Humans and Other Species: Emerging Candidate Tumour Markers૾
    Clinical Biochemistry 36 (2003) 443–452 An overview of the kallikrein gene families in humans and other species: Emerging candidate tumour markers૾ George M. Yousefa,b, Eleftherios P. Diamandisa,b,* aDepartment of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Abstract Kallikreins are serine proteases with diverse physiologic functions. They are represented by multigene families in many animal species, especially in rat and mouse. Recently, the human kallikrein gene family has been fully characterized and includes 15 members, tandemly localized on chromosome 19q13.4. A new definition has now been proposed for kallikreins, which is not based on function but, rather, on close proximity and structural similarities. In this review, we summarize available information about kallikreins in many animal species with special emphasis on human kallikreins. We discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and overview their evolutionary history. Kallikreins are expressed in a wide range of tissues including the salivary gland, endocrine or endocrine-related tissues such as testis, prostate, breast and endometrium and in the central nervous system. Most, if not all, genes are under steroid hormone regulation. Accumulating evidence indicates that kallikreins are involved in many pathologic conditions. Of special interest is the potential role of kallikreins in the central nervous system. In addition, many kallikreins seem to be candidate tumor markers for many malignancies, especially those of endocrine-related organs. © 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Kallikrein; Tumor markers; Cancer biomarkers; Prostate cancer; Breast cancer; Ovarian cancer; Alzheimer’s disease; Serine proteases; Chromosome 19; Kallikrein evolution; Rodent kallikreins; Hormonally regulated genes 1.
    [Show full text]
  • AACR2006-1686P
    Funding Proteinases and Inflammation Network Group grant Kallikrein 14 Signalling through Proteinase-Activated Receptors CIHR operating grant 1,2 3,4 3,4 3,4 5 Alberta Heritage Foundation for Katerina Oikonomopoulou , Kristina K. Hansen , Mahmoud Saifeddine , Illa Tea , Patricia Andrade-Gordon , Medical Research Graeme S. Cottrell6, Nigel W. Bunnett6, Nathalie Vergnolle3, Eleftherios P. Diamandis1,2 and Morley D. Hollenberg3,4 NSERC / CRSNG 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto; 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto; 3Department of Pharmacology & Therapeutics, and 4Department of Medicine, University of Calgary, Calgary; 5Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania, 6Departments of Surgery and Physiology, University of California, San Francisco Kallikrein 14 can selectively disarm PAR (lower concentrations), OVERVIEW Figure 1: Mechanism of PAR activation (activation by proteolysis) 2. Kallikrein 14 can selectively disarm PAR1 (lower concentrations), 2+ Proteinase-activated receptors (PARs): a family of G-protein coupled receptors activated by serine proteinase cleavage site whilst activating PARs 2 and 4; Ca in HEK cells proteinases via a proteolytically revealed ‘tethered ligand’ (Fig. 1). Four family members (Fig. 2); PARs N PAR disarming 1, 2 and 4 signal to cells. N PAR1 dis-arming1 vs. activation Selective activation of PAR4 vs. PARs 1, 2 Human kallikreins (hKs): A 15-member family of secreted serine proteinases implicated in tumour 1 cm Thrombin 1 cm 2 min 2 min TFLLR-NH2 progression and cell survival (Fig. 4). (selective desensitization hK14: a tryptic kallikrein; wide tissue distribution, implicated in breast and ovarian cancer (Fig. 5 and 6). of PAR1) The mechanism of kallikrein action is not yet known: Although some targets have been identified (e.g.
    [Show full text]
  • Solenodon Genome Reveals Convergent Evolution of Venom in Eulipotyphlan Mammals
    Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals Nicholas R. Casewella,1, Daniel Petrasb,c, Daren C. Cardd,e,f, Vivek Suranseg, Alexis M. Mychajliwh,i,j, David Richardsk,l, Ivan Koludarovm, Laura-Oana Albulescua, Julien Slagboomn, Benjamin-Florian Hempelb, Neville M. Ngumk, Rosalind J. Kennerleyo, Jorge L. Broccap, Gareth Whiteleya, Robert A. Harrisona, Fiona M. S. Boltona, Jordan Debonoq, Freek J. Vonkr, Jessica Alföldis, Jeremy Johnsons, Elinor K. Karlssons,t, Kerstin Lindblad-Tohs,u, Ian R. Mellork, Roderich D. Süssmuthb, Bryan G. Fryq, Sanjaya Kuruppuv,w, Wayne C. Hodgsonv, Jeroen Kooln, Todd A. Castoed, Ian Barnesx, Kartik Sunagarg, Eivind A. B. Undheimy,z,aa, and Samuel T. Turveybb aCentre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA Liverpool, United Kingdom; bInstitut für Chemie, Technische Universität Berlin, 10623 Berlin, Germany; cCollaborative Mass Spectrometry Innovation Center, University of California, San Diego, La Jolla, CA 92093; dDepartment of Biology, University of Texas at Arlington, Arlington, TX 76010; eDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; fMuseum of Comparative Zoology, Harvard University, Cambridge, MA 02138; gEvolutionary Venomics Lab, Centre for Ecological Sciences, Indian Institute of Science, 560012 Bangalore, India; hDepartment of Biology, Stanford University, Stanford, CA 94305; iDepartment of Rancho La Brea, Natural History Museum of Los Angeles County, Los Angeles,
    [Show full text]
  • Aberrant Human Tissue Kallikrein Levels in the Stratum Corneum and Serum of Patients with Psoriasis: Dependence on Phenotype, Severity and Therapy N
    CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j.1365-2133.2006.07743.x Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy N. Komatsu,* à K. Saijoh,§ C. Kuk,* F. Shirasaki,à K. Takeharaà and E.P. Diamandis* *Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada àDepartment of Dermatology and §Department of Hygiene, Graduate School of Medical Science, School of Medicine, Kanazawa University, Kanazawa, Japan Summary Correspondence Background Human tissue kallikreins (KLKs) are a family of 15 trypsin-like or Eleftherios P. Diamandis. chymotrypsin-like secreted serine proteases (KLK1–KLK15). Multiple KLKs have E-mail: [email protected] been quantitatively identified in normal stratum corneum (SC) and sweat as can- didate desquamation-related proteases. Accepted for publication 7 November 2006 Objectives To quantify KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of patients with psoriasis, and their variation Key words between lesional and nonlesional areas and with phenotype, therapy and severity. diagnostic marker, human kallikreins, psoriasis, The overall SC serine protease activities were also measured. serine proteases, stratum corneum, therapy Methods Enzyme-linked immunosorbent assays and enzymatic assays were used. Conflicts of interest Results The lesional SC of psoriasis generally contained significantly higher levels None declared. of all KLKs. KLK6, KLK10 and KLK13 levels were significantly elevated even in the nonlesional SC. The overall trypsin-like, plasmin-like and furin-like activities were significantly elevated in the lesional SC.
    [Show full text]
  • KLK15 Is a Prognostic Marker for Progression?
    IJC International Journal of Cancer KLK15 is a prognostic marker for progression-free survival in patients with radical prostatectomy Anja Rabien1, Florian R. Fritzsche2,3, Monika Jung1, Angelika To¨lle1, Eleftherios P. Diamandis4, Kurt Miller1, Klaus Jung1,5, Glen Kristiansen2,3y and Carsten Stephan1,3y 1 Department of Urology, Charite´ - Universita¨tsmedizin Berlin, Campus Charite´ Mitte, Berlin, Germany 2 Institute of Pathology, Charite´ - Universita¨tsmedizin Berlin, Campus Charite´ Mitte, Berlin, Germany 3 Institute of Pathology, Universita¨tsspital Zu¨rich, Zu¨rich, Switzerland 4 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 5 Berlin Institute for Urologic Research, Berlin, Germany In search of biomarkers for prostate cancer, we evaluated the expression of the human kallikrein-related peptidase KLK15 in samples of prostatic adenocarcinomas from radical prostatectomies. Twenty-five pairs of cancerous and adjacent normal prostatic tissue were selected by laser capture microdissection. The tissue was used for quantification of KLK15 mRNA by reverse-transcriptase polymerase chain reaction. Immunohistochemical expression of the KLK15 protein in 193 samples of prostatic adenocarcinoma was analysed in relation to clinicopathological parameters of the patients and disease progression. Expression of KLK15 correlated with the pathological tumour stage and Gleason score of the cases, both at mRNA and at protein level. While mRNA expression in the tumour was elevated, the protein level of KLK15 was reduced compared with adjacent normal tissue and to prostatic intraepithelial neoplasia. Univariate Kaplan-Meier analysis showed a significant association of dichotomised KLK15 levels with disease progression defined by prostate-specific antigen relapse (p 5 0.001).
    [Show full text]
  • Human Tissue Kallikrein Expression in the Stratum Corneum and Serum of Atopic Dermatitis Patients
    DOI:10.1111/j.1600-0625.2007.00562.x www.blackwellpublishing.com/EXD Original Article Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients Nahoko Komatsu1,2,3,4, Kiyofumi Saijoh4, Cynthia Kuk1, Amber C. Liu1, Saba Khan1, Fumiaki Shirasaki3, Kazuhiko Takehara3 and Eleftherios P. Diamandis1,2 1Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; 3Department of Dermatology, Graduate School of Medical Science, School of Medicine, Kanazawa University, Kanazawa, Japan; 4Department of Hygiene, Graduate School of Medical Science, School of Medicine, Kanazawa University, Kanazawa, Japan Correspondence: Eleftherios P. Diamandis, MD, PhD, FRCPC, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada, Tel.: +1 416 586 8443, Fax: +1 416 586 8628, e-mail: [email protected] Accepted for publication 9 March 2007 Abstract: Human tissue kallikreins are a family of 15 trypsin- or differ significantly. In the serum of AD patients, KLK8 was chymotrypsin-like secreted serine proteases (KLK1–KLK15). Many significantly elevated and KLK5 and KLK11 were significantly KLKs have been identified in normal stratum corneum (SC) and decreased. However, their serum levels were not modified by sweat, and are candidate desquamation-related proteases. We corticosteroid topical agents. The alterations of KLK levels in the report quantification by enzyme-linked immunosorbent assay SC of AD were more pronounced than those in the serum. KLK7 (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and in the serum was significantly correlated with eosinophil counts in KLK14 in the SC and serum of atopic dermatitis (AD) patients by the blood of AD patients, while KLK5, KLK8 and KLK11 were ELISA, and examine their variation with clinical phenotype, significantly correlated with LDH in the serum.
    [Show full text]
  • Supplementary Material Contents
    Supplementary Material Contents Immune modulating proteins identified from exosomal samples.....................................................................2 Figure S1: Overlap between exosomal and soluble proteomes.................................................................................... 4 Bacterial strains:..............................................................................................................................................4 Figure S2: Variability between subjects of effects of exosomes on BL21-lux growth.................................................... 5 Figure S3: Early effects of exosomes on growth of BL21 E. coli .................................................................................... 5 Figure S4: Exosomal Lysis............................................................................................................................................ 6 Figure S5: Effect of pH on exosomal action.................................................................................................................. 7 Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant ....... 8 Effective exosomal concentration....................................................................................................................8 Figure S7: Sample constitution for luminometry experiments..................................................................................... 8 Figure S8: Determining effective concentration .........................................................................................................
    [Show full text]
  • Purification and Characterization of Human Kallikrein 11, a Candidate Prostate and Ovarian Cancer Biomarker, from Seminal Plasma Liu-Ying Luo,1, 2 Shannon J.C
    Human Cancer Biology Purification and Characterization of Human Kallikrein 11, a Candidate Prostate and Ovarian Cancer Biomarker, from Seminal Plasma Liu-Ying Luo,1, 2 Shannon J.C. Shan,1, 2 Marc B. Elliott,1, 2 Antoninus Soosaipillai,1 and Eleftherios P. Diamandis1, 2 Abstract Purpose: Preliminary data suggest that hK11is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11from seminal plasma. Experimental Design: hK11was purified from seminal plasma by immunoaffinity chromatogra- phy and characterized by kinetic analysis, electrophoresis,Western blots, and mass spectrometry. Results: hK11is present in seminal plasma at concentrations ranging from 2 to 37 Ag/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11to homogeneity. In seminal plasma, hK11is present as a free enzyme of f40 kDa. About 40% of hK11is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg15 6 (Genbank accession no. AF164623), which generates two peptides of f20 kDa, connected by internal disulfide bonds. Purified hK11possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, a1-antichymotrypsin, a2-antiplasmin, and a1-antitrypsin have no effect on hK11activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11- specific monoclonal antibody inhibited hK11activity up to 40%. Plasmin is a strong candidate for cleaving hK11at Arg15 6. Conclusion: This is the first report on purification and characterization of native hK11.We speculate that hK11,along with other kallikreins, proteases, andinhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.
    [Show full text]
  • Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases Hyesook Yoon
    Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2008 Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases Hyesook Yoon Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ACTIVATION PROFILES AND REGULATORY CASCADES OF THE HUMAN KALLIKREIN-RELATED PEPTIDASES By HYESOOK YOON A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2008 The members of the Committee approve the dissertation of Hyesook Yoon defended on July 10th, 2008. ________________________ Michael Blaber Professor Directing Dissertation ________________________ Hengli Tang Outside Committee Member ________________________ Brian Miller Committee Member ________________________ Oliver Steinbock Committee Member Approved: ____________________________________________________________ Joseph B. Schlenoff, Chair, Department of Chemistry and Biochemistry The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGMENTS I would like to dedicate this dissertation to my parents for all your support, and my sister and brother. I would also like to give great thank my advisor, Dr. Blaber for his patience, guidance. Without him, I could never make this achievement. I would like to thank to all the members in Blaber lab. They are just like family to me and I deeply appreciate their kindness, consideration and supports. I specially like to thank to Mrs. Sachiko Blaber for her endless guidance and encouragement. I would like to thank Dr Jihun Lee, Margaret Seavy, Rani and Doris Terry for helpful discussions and supports.
    [Show full text]
  • WO 2019/046815 Al 07 March 2019 (07.03.2019) W 1P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/046815 Al 07 March 2019 (07.03.2019) W 1P O PCT (51) International Patent Classification: OSTERTAG, Eric [US/US]; 4242 Campus Point Court, C12N 15/90 (2006.01) Suite 700, San Diego, California 82121 (US). RICHTER, Maximilian [US/US]; 473 1Kansas Street, San Diego, Cal¬ (21) International Application Number: ifornia 921 16 (US). CRANERT, Stacey Ann [US/US]; PCT/US20 18/049257 7693 Palmilla Dr. Apt. 2103, San Diego, California 92122 (22) International Filing Date: (US). 31 August 2018 (3 1.08.2018) (74) Agent: MILLER, Katherine J. et al.; COOLEY LLP, (25) Filing Language: English 1299 Pennsylvania Avenue, NW, Suite 700, Washington, District of Columbia 20004 (US). (26) Publication Language: English (81) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of national protection available): AE, AG, AL, AM, 62/552,861 31 August 2017 (3 1.08.2017) US AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 62/558,286 13 September 2017 (13.09.2017) US CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/608,546 20 December 2017 (20. 12.2017) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (71) Applicant: POSEIDA THERAPEUTICS, INC. HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, [US/US]; 4242 Campus Point Court, Suite 700, San Diego, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, California 92121 (US).
    [Show full text]
  • A Kallikrein 15 (KLK15) Single Nucleotide Polymorphism Located
    Batra et al. BMC Cancer 2011, 11:119 http://www.biomedcentral.com/1471-2407/11/119 RESEARCHARTICLE Open Access A Kallikrein 15 (KLK15) single nucleotide polymorphism located close to a novel exon shows evidence of association with poor ovarian cancer survival Jyotsna Batra1,2, Christina M Nagle3, Tracy O’Mara1,2, Melanie Higgins1,2, Ying Dong1, Olivia L Tan1, Felicity Lose2, Lene Marie Skeie1, Srilakshmi Srinivasan1, Kelly L Bolton4,5, Honglin Song4, Susan J Ramus6, Simon A Gayther6, Paul DP Pharoah4, Mary-Anne Kedda1, Amanda B Spurdle2 and Judith A Clements1* Abstract Background: KLK15 over-expression is reported to be a significant predictor of reduced progression-free survival and overall survival in ovarian cancer. Our aim was to analyse the KLK15 gene for putative functional single nucleotide polymorphisms (SNPs) and assess the association of these and KLK15 HapMap tag SNPs with ovarian cancer survival. Results: In silico analysis was performed to identify KLK15 regulatory elements and to classify potentially functional SNPs in these regions. After SNP validation and identification by DNA sequencing of ovarian cancer cell lines and aggressive ovarian cancer patients, 9 SNPs were shortlisted and genotyped using the Sequenom iPLEX Mass Array platform in a cohort of Australian ovarian cancer patients (N = 319). In the Australian dataset we observed significantly worse survival for the KLK15 rs266851 SNP in a dominant model (Hazard Ratio (HR) 1.42, 95% CI 1.02- 1.96). This association was observed in the same direction in two independent datasets, with a combined HR for the three studies of 1.16 (1.00-1.34).
    [Show full text]