Seprase; an overview of an important matrix serine protease Pamela O’Brien a *and Brendan F. O’Connor a b a School of Biotechnology, Dublin City University, Dublin 9, Ireland. b Centre for BioAnalytical Sciences, Dublin City University, Dublin 9, Ireland. * CORRESPONDENCE * Corresponding author for proofs: Pamela O’Brien, School Of Biotechnology, Dublin City University, Dublin 9, Ireland. Email address: [email protected] Phone: +353-1-7005908 Fax: +353-1-7005412 KEYWORDS (6) Seprase; Fibroblast Activation Protein alpha; Serine peptidase; Antiplasmin Cleaving Enzyme; Serine Integral Membrane Protein ABBREVIATIONS AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; AFC, 7-amino-4- trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; APSF, 4-amidino phenylsulfonyl fluoride; DABCYL, 4-(4-dimethylaminophenylazo)benzoyl; DFP, Diisopropyl fluorophosphates; DPPIV, Dipeptidyl peptidase IV; DTT, Dithiothreitol; ECM, Extracellular matrix; EDANS, 5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid; EDTA, Ethylenediaminetetra acetic acid; FGF-2, Fibroblast growth factor-2; GBase, Guanidinobutyrase; MMP, Matrix metallo-proteinase; mAb, monoclonal antibody; NEM, N-ethylmaleimide; PMSF, Phenylmethylsulphonyl fluoride; POP, Prolyl Oligopeptidase; SIMP, Serine Integral Membrane Protein; uPA, Urokinase plasminogen activator - 1 - Abstract Seprase or Fibroblast activation protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which include dipeptidyl peptidase IV (DPPIV/CD26) and related type II trans-membrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localization of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis. - 2 - 1. Introduction Wound healing and tumour development are dynamic progressive processes that involve the interaction of several tissue types and have many mechanistic similarities. The composition of tumour stroma markedly resembles that of wound granulation tissue, although a distinguishing feature of the tumour stroma is the absence of platelets and a lower density of inflammatory cells [1]. In cancer, these changes in the stroma drive invasion and metastasis, the hallmarks of malignancy (Fig. 1.). Tumour cells can produce many of the same growth factors that activate the adjacent stromal tissues as in wounding or fibrosis. Activated fibroblasts and infiltrating immune cells (macrophages) secrete proteases (MMPs) and cytokines such as Fibroblast Growth Factor-2 (FGF-2). These factors potentiate tumour growth, stimulate angiogenesis and induce fibroblasts to undergo differentiation into myofibroblasts and into smooth muscle [2]. Cell surface proteases play an important role in facilitating cell invasion into the extracellular matrix. Proteases associate at plasma membrane protrusions, called invadopodia, which contact and dissolve the matrix. Invadopodia degrade a variety of immobilised substrates including fibronectin, laminin and type I collagen [3]. Integral membrane proteases may contribute significantly to ECM degradation by metastatic cells by virtue of their localisation at invadopodia which are in contact with the ECM. Integral membrane proteases can be defined as a group of cell surface glycoproteins that contain extracellular domains of either metallo- or serine-proteases, a transmembrane domain and a short cytoplasmic tail. Examples of such transmembrane glycoproteins include meprin, matrix metalloproteinase, DPPIV, - 3 - fibroblast activation protein α (FAPα) / Seprase and GBase. Abundant expression of these enzymes is associated with poor prognosis [4]. This review will take a detailed look at the serine protease Seprase (surface expressed protease) or Fibroblast Activation Protein α (FAPα), a 170kDa integral membrane gelatinase, which belongs to the S9b peptidase family [5, 6]. FAPα, formerly known as F19 Cell Surface Antigen, is an inducible cell surface glycoprotein that was originally identified in 1986 in cultured fibroblasts using the monoclonal antibody (mAb) F19 [7-9][7, 8]. In the early years using the mAb F19, malignant epithelial cancers such as breast, lung, colon and ovarian were found to be F19- . In 1994, the F19 Cell Surface Antigen was named Fibroblast Activation Protein α (FAPα) . In 1990 a 170kDa gelatinase was identified in the human malignant melanoma cell line LOX . The 170kDa membrane-bound protease was found to be associated with the expression of invasiveness by human malignant melanoma cells. In 1994, the 170kDa gelatinase was named Seprase . Seprase was originally identified as a glycoprotein peptidase selectively expressed on the surface of invadopodia and was isolated from a human malignant melanoma cell line LOX [3, 12][9]. The expression of Seprase correlates with the invasiveness of human melanoma and carcinoma cells [12][9]. Using anti-Seprase mAbs, Seprase was seen to be expressed in stromal fibroblasts of more than 90% of all epithelial tumours including lung, colorectal and breast carcinomas (primary and metastatic) . Molecular cloning of both FAPα and Seprase revealed that they are the same cell surface serine protease which is found on chromosome 2q23 [12-16][9-13]. The fact that FAPα and Seprase are the same protease means that the above immunohistochemistry results contradict each other. The reason for the differing results is due to the different monoclonal antibodies - 4 - recognising different parts of the serine protease. For the clarity of this review, the protease is referred to as Seprase throughout. 2. Classification of Seprase / Fibroblast Activation Protein alpha (FAPα) Seprase (EC 3.4.21.B28) belongs to the small family of serine integral membrane peptidases (SIMPs). These peptidases are inducible, specific for proline-containing peptides and macromolecules and active on the cell surface [15]. Post proline peptidases modify bioactive peptides and change their cellular functions. This class of peptidases have important roles in cancer [14, 15, 17-19]. This group of enzymes also includes prolyl endopeptidase, dipeptidyl peptidase 8 and dipeptidyl peptidase IV-β [19]. However, the best studied of this class of enzymes is dipeptidyl peptidase IV (DPPIV or CD26) (EC 3.4.14.5) [19]. Studies have shown the importance of DPPIV in regulating tumour cell behaviour and function [20]. Seprase shows up to 52% homology to DPPIV, both being members of the S9b peptidase family [21]. Seprase is, therefore, a member of the DPPIV-like gene family [5] grouped in the subfamily S9b of the peptidase family S9 (prolyl oligopeptidase family), clan SC [6]. Even though all SIMP members are known to cleave prolyl peptide (Pro-Xaa) bonds there are conflicting reports on possible dipeptidyl peptidase activity associated with Seprase but its main distinguishing feature is its gelatinase activity [22, 23]. An early report had suggested that DPPIV had gelatinase activity [24]. However, more recent reports suggest otherwise [22, 23]. The exact nature of the physiological roles Seprase plays are only beginning to be understood but insights into potential functions of Seprase can be obtained from the vast amount of work done on DPPIV. - 5 - - 6 - 3. Structure and Biochemical Aspects Active Seprase is a 170kDa homodimer that contains two N-glycosylated 97kDa subunits. The 760 amino acid Seprase protein (GenBank GI 1888316) is a type II integral membrane protein with a large C-terminal extracellular domain. Seprase has been shown to shed from the cell surface and recent studies by our group have identified a serum form of the protease [25][22]. A second group has recently identified the soluble form of Antiplasmin Cleaving Enzyme (APCE) as Seprase [26][23]. The Seprase monomer has 5 potential N-glycosylation sites, 13 cysteine residues, 3 segments that correspond to highly conserved catalytic domains of serine proteases, a hydrophobic transmembrane segment and a short cytoplasmic tail (6 amino acids) (Fig. 1. ref [14][11]). The crystal structure of the extracellular domain of Seprase has recently been resolved (Fig. 2.) [27][24]. This has provided valuable information on the substrate specificity of Seprase (discussed further in this Section and also in Section 7.). Each subunit contains topologically distinct domains: the β-propeller (residues 54- 492) and the α/β-hydrolase domain (residues 27-53 and 493-760) (Fig. 2.). The catalytic triad is located at the interface of the β-propeller and the α/β-hydrolase domain. The arrangement of the catalytic triad in the order nucleophile-acid-base is a characteristic of the α/β hydrolase domain [28][25]. This domain features mostly parallel β-sheets connected by α-helices on either surface of the sheet (Fig. 3A.). The sheets are twisted and radially arranged around their ventral