Available online at www.sciencedirect.com
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`
`Biochimica et Biophysica Acta 1784 (2008) 1130 – 1145
`
`Bioch{m lc a et 8. BSA
`
`www.elsevier.com/locate/bbapap
`
`Review
`Seprase: An overview of an important matrix serine protease
`Pamela O'Brien a,⁎, 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
`
`Received 3 October 2007; received in revised form 9 January 2008; accepted 10 January 2008
`Available online 26 January 2008
`
`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 includes dipeptidyl peptidase IV (DPPIV/CD26) and related type
`II transmembrane 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. Localisation 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.
`© 2008 Elsevier B.V. All rights reserved.
`
`Keywords: Seprase; Fibroblast Activation Protein α; Serine peptidase; Antiplasmin Cleaving Enzyme; Serine integral membrane protein
`
`1. Introduction
`
`Wound healing and tumour development are dynamic pro-
`gressive 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
`
`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, Phenylmethylsulfonyl fluoride; POP, Prolyl oligopeptidase; SIMP,
`Serine Integral Membrane Protein; uPA, Urokinase plasminogen activator
`⁎ Corresponding author. Tel.: +353 1 7005908; fax: +353 1 7005412.
`E-mail address: pamelaobrien4@gmail.com (P. O'Brien).
`
`1570-9639/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
`doi:10.1016/j.bbapap.2008.01.006
`
`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 metallo-proteinase,
`
`Petitioner GE Healthcare – Ex. 1025, p. 1130
`
`

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`P. O'Brien, B.F. O'Connor / Biochimica et Biophysica Acta 1784 (2008) 1130–1145
`
`1131
`
`Tissue Integrity
`
`D
`
`Normal Epithelium
`
`_
`
`P_1_~ _r _I------- ~
`~ __ c.nc.r ____ ~I ~------------~ I Cancer Stroma
`
`D $
`
`Fig. 1. Summary of intracellular signalling. Signalling from the normal epithelial
`cells to the stroma and back (black thin arrows) maintains the integrity of the
`epithelial tissue (thick arrow). During epithelial carcinogenesis, the signalling
`changes (dotted blue arrow) and this causes cancer-associated changes in the
`stroma. The new cross-talk (dotted black arrows) between the cancer cells and the
`stroma leads to invasion (thick arrow). Adapted from De Wever and Mareel
`[112], and used with permission.
`
`DPPIV, 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 170 kDa 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]. In
`the early years using the mAb F19, malignant epithelial cancers
`such as breast, lung, colon and ovarian were found to be F19−
`[9]. In 1994, the F19 Cell Surface Antigen was named Fibroblast
`Activation Protein α (FAPα) [10]. In 1990 a 170 kDa gelatinase
`was identified in the human malignant melanoma cell line LOX
`[11]. The 170 kDa membrane-bound protease was found to be
`associated with the expression of invasiveness by human malig-
`nant melanoma cells. In 1994, the 170 kDa gelatinase was
`named Seprase [3]. Seprase was 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]. The expression of Seprase correlates with the
`invasiveness of human melanoma and carcinoma cells [12].
`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) [7]. 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]. The fact that FAPα and
`Seprase are the same protease means that the above immuno-
`histochemistry results contradict each other. The reason for the
`
`differing results is due to the different monoclonal antibodies
`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 [17–19]. This group of enzymes also includes prolyl
`endopeptidase, dipeptidyl peptidase 8 and dipeptidyl peptidase
`IV-β [14,15,19]. 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.
`
`3. Structure and biochemical aspects
`
`Active Seprase is a 170 kDa homodimer that contains two
`N-glycosylated 97 kDa 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].
`A second group has recently identified the soluble form of
`Antiplasmin Cleaving Enzyme (APCE) as Seprase [26].
`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 of Ref. [14]). The crystal structure of the extra-
`cellular domain of Seprase has recently been resolved (Fig. 2)
`[27]. 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
`
`Petitioner GE Healthcare – Ex. 1025, p. 1131
`
`

`

`1132
`
`P. O'Brien, B.F. O'Connor / Biochimica et Biophysica Acta 1784 (2008) 1130–1145
`
`
`
`C a f ' 1 1 J t i c w p H y d r o / a s < '
`D o m a i n ( £ : r e e n )
`/ \
`
`\
`8 B l a d e d P· p r o p e l / e r
`( b l u e )
`
`Fig. 2. Three-dimensional structure of Seprase. The ribbon diagram illustrates the dimeric structure of Seprase (pdb accession code 1Z68). The extracellular domain
`consists of 2 domains, an eight bladed β-propeller domain (indicated in blue) and an α/β hydrolase domain (indicated in green) that contains the catalytic triad. The
`catalytic residues are shown, catalytic Serine 624 (purple), Aspartic Acid 702 (cyan) and Histidine 734 (yellow). Generated using DeepView [113].
`
`domain. The arrangement of the catalytic triad in the order
`nucleophile–acid–base is a characteristic of the α/β hydrolase
`domain [28]. 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
`tunnel. The eight bladed β-propeller domain is situated on top
`of the catalytic triad and may serve as a ‘gate’ to selectively
`filter protein access to the catalytic triad (Fig. 3B) [27,29]. The
`β-propeller domain in prolyl oligopeptidase has been shown to
`regulate proteolysis [30]. The oscillating propeller blades have
`been shown to act as a gating filter during catalysis, letting small
`peptide substrates into the active site while excluding large
`proteins to prevent accidental proteolysis in the cytosol. The
`active site is accessible in two ways: (i) through a cavity formed
`between the β-propeller and (ii) through the hydrolase domain.
`The side opening has a diameter of ∼24 Å in contrast to the
`narrower β-propeller opening (∼14 Å).
`As mentioned earlier, DPPIV shows similar structure
`homology to Seprase. In DPPIV, the N-terminal hydrophobic
`sequence represents an uncleavable signal peptide, that also
`functions as a membrane-anchoring domain [6,31]. In Seprase,
`the N-terminal domain possibly has a similar role as a signal
`peptide, although there is no published data to support this. A
`highly conserved residue Asp599 in DPPIV has been shown to
`be important in enzyme processing such as proper folding,
`dimerisation and transport [15]. A mutation in this residue
`(D599A) specifically decreased the cell surface expression of
`DPPIV in stably transfected mouse fibroblasts. Seprase also has
`this conserved Asp599 residue (Fig. 4); therefore, it is possible to
`
`conclude that this residue is also important in the processing of
`the Seprase enzyme.
`The Seprase gene has been observed in several species
`(Fig. 4). A mouse homologue has been identified [32] as well as
`a Xenopus laevis homologue [33]. The mouse Seprase gene
`spans approximately 60 kb and contains 26 exons ranging in
`size from 46 bp to 195 bp. This genomic organisation is similar
`to that of the human Seprase gene [32]. The catalytic serine
`residue arranged within the consensus sequence G-X-S-X-G is
`split between two exons. Gly-Trp is located at the very end of
`exon 21 and Ser-Tyr-Gly at the beginning of exon 22 (discussed
`further in Section 3.1). This arrangement differs from the typical
`serine protease where the complete serine consensus site is
`encoded within one exon. The study of the mouse homologue
`has shown alternative splicing and 3 distinct Seprase splice
`variants have been detected in tissues [34]. An alternative
`spliced Seprase was later identified in the human melanoma cell
`line LOX which encodes a novel truncated isoform [35]. The
`splice variant encodes for a 239 amino acid polypeptide with a
`molecular weight of 27 kDa that precisely overlaps the carboxyl-
`terminal catalytic region of the wild type Seprase. An alignment
`of eukaryotic Seprase C-terminal amino acid sequences is shown
`in Fig. 4. Sequence homology is represented in grey scale
`shading, with black being the highest homology. This figure also
`illustrates that the C-terminal catalytic region of Seprase is
`highly homologous throughout the different species.
`The charged N-terminal end of substrate peptides is recog-
`nised by two glutamates (Glu motif). Comparison of the crystal
`structure of Seprase and DPPIV revealed one major difference in
`
`Petitioner GE Healthcare – Ex. 1025, p. 1132
`
`

`

`P. O'Brien, B.F. O'Connor / Biochimica et Biophysica Acta 1784 (2008) 1130–1145
`
`1133
`
`Structural composition of the active sites of Seprase and
`DPPIV revealed similar S2–S2′ specificity pockets. The S1′
`subsite (numbered according to [37]) in Seprase is flat and could
`accommodate most amino acids. The S2′ active site pocket is
`lined by Trp623 and Tyr745. These residues would be expected to
`interact with large aliphatic side chains of peptide substrates.
`The S1 specificity pocket in Seprase is a well defined hydro-
`phobic pocket lined by Tyr625, Val650, Trp653, Tyr656, Tyr660
`and Val705. This site optimally accommodates a proline residue.
`Large hydrophobic and aromatic residues can be modelled in
`the hydrophobic S2 pocket, defined by residues Arg123, Phe350,
`Phe351, Tyr541, Pro544, Tyr625 and Tyr660 [27].
`
`3.1. Catalytic classification
`
`In its membrane form, the majority of Seprase including its
`catalytic domain is exposed to the extracellular environment
`(Fig. 1 of Ref. [14]). The catalytic domain consists of the
`catalytic serine (S624) flanked by glycines in the classical
`consensus sequence for an active site serine, G-X-S-X-G. This
`conserved serine protease motif is present as G-W-S-Y-G. The
`catalytic serine in conjunction with Asp702 and His734 com-
`prises the catalytic triad [12,16,21]. The orientation of these
`residues is similar to members of the prolyl oligopeptidase
`family and its structural organisation is similar to that of DPPIV.
`Therefore, this enzyme is classified as a non-classical serine
`protease. An interesting observation made in DPPIV is that a
`single substitution of either Gly residue in the motif resulted in
`the retention of the newly synthesised enzyme in the endo-
`plasmic reticulum and rapid degradation [6]. This suggests that
`both residues are also essential for correct folding of the enzyme
`and transport to the cell surface. The histidine acts as a general
`acid–base catalyst activating the nucleophilic group,
`the
`hydroxyl group of the serine acts as a nucleophile in the attack
`on the peptide bond while the aspartic acid stabilises charged
`tetrahedral intermediates formed in the reaction [38,39].
`
`3.2. Biochemical properties of Seprase
`
`Post translational modifications of the Seprase protein such
`as N-glycosylation occur and it is thought that the N-terminus
`may be blocked [12]. The resolved crystal structure of Seprase
`shows that there are 5 potential glycosylation sites on the
`asparagine residues 49, 92, 227, 314 and 679. Four are located
`in the β-propeller domain and one is located in the hydrolase
`domain [27]. The glycosylated form of Seprase has both post-
`prolyl dipeptidyl peptidase and gelatinase activities while the
`non-glycosylated form lacks any detectable activity [40].
`Reports show that the gelatinase activity of Seprase was
`completely blocked by serine protease inhibitors, including
`DFP and PMSF [11,25]. Seprase could be affinity labelled by
`[3H]-DFP, but the proteolytically inactive 97 kDa subunit could
`not be [12]. This confirmed the existence of a serine protease
`active site on the dimeric form of the enzyme. This was further
`demonstrated by the loss of proteolytic activity upon the
`dissociation of its 97 kDa subunits following treatment with
`acid, heat, or cysteine and histidine modifying agents [12].
`
`8
`
`S i n g l e f l - p r o p e l l e r
`B l a d e ( t r i a n g l e )
`
`Fig. 3. Three-dimensional Seprase domains. (A) The ribbon diagram illustrates
`the α/β hydrolase domain (residues 27–53 and 493–760) of Seprase (pdb
`accession code 1Z68). The α/β hydrolase domain contains the catalytic triad.
`The catalytic residues are shown, catalytic Serine 624 (pink), Aspartic Acid 702
`(cyan) and Histidine 734 (yellow). α-helices are indicated in red, β-sheets are
`indicated in blue and the hydrogen bonds are indicated in green. (B) The ribbon
`diagram illustrates the eight bladed β-propeller domain (residues 54–492) of
`Seprase (pdb accession code 1Z68). Generated using DeepView [113].
`
`the vicinity of the Glu motif (Glu203-Glu204 for Seprase; Glu205-
`Glu206 for DPPIV) within the active site of the enzyme (Fig. 2 of
`Ref. [27]). The importance of the Glu motif in DPPIV catalysis
`has been confirmed by single-point mutations that abolish the
`enzymes aminopeptidase activity [36]. Detailed comparison of
`Seprase and DPPIV revealed that the Ala657 residue in Seprase,
`instead of Asp663 as in DPPIV, reduces the acidity in this pocket.
`This change could explain the lower affinity for N-terminal
`amines by Seprase [27]. Mutant proteins were developed to
`determine the importance of these two residues. Studies
`have shown that in DPPIV, the replacement of Asp663 by an
`Ala663 results in a ∼4-fold decrease in catalytic efficiency for
`N-terminal dipeptides, with a concomitant increase in efficiency
`to cleave Z-Gly-Pro-AMC (which has been shown to be cleaved
`by DPPIV). This mutation caused the wild type DPPIV catalytic
`efficiency for Z-Gly-Pro-AMC to be increased from 9 M− 1 s− 1
`to 1.6 M− 1 s− 1.
`
`Petitioner GE Healthcare – Ex. 1025, p. 1133
`
`

`

`1134
`
`P. O'Brien, B.F. O'Connor / Biochimica et Biophysica Acta 1784 (2008) 1130–1145
`
`H. sapiens
`H. m.usculu
`X. laevis
`B . "t.aurus
`
`H. sapiens
`H . :ra.uscul u
`X . laevis
`B. taurus
`
`H. sapiens
`H. m.uscul u
`X . laevis
`B . taurus
`
`*
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`FIJll!GFIDEmRIAI1JJG1JJSYGGY'JSSLAL1'SCTCLPRCGIAVAPVS$1JEYYASIY9ERF!IGLPTl(D)mL~HYKUST'Jl!.>:R.'.EYFPNVDYLLIHGTADDMVHF
`Fit!GFIDE~Rl.'.I1JJG1JJSYGGYVf!s1t1~cc;,Dpvs;EullYYASIYTllRYl!GLPT$llLFJ"f.
`ISTV!itJR.,.EmF~YLLIIHGTADDMVHF
`Fil!l!GFIDEKRI.'.I1JJG1JJSYGGY'JSSLALASCTCLFKCGIA\IAPVS$1JllYY.'.SI\~llRFUGLPTl(DDJJ!J
`IST'Jl!P'.R.>.Ei.'FPlfJDYLLIHGTADDMVHF
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`*
`
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`sos
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`ll6
`
`606
`606
`603
`217
`
`707
`707
`704
`318
`
`H . sapiens
`H. m.usculu
`X . laevis
`B . eaurus
`
`QJ,J'~Q IllU.L 'J!IAQVD F QPJThlY~DQIIH0
`QMSAQ rk~ L 'Jl,r AQVD F QPJThlYSDd11H~
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`aa f~ar~.LVB AQVDFQ>JruYIJo~D
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`
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`S~ffiLFTmlTHFL!mmMDL
`LSGLSTI ffiLYTmJTHFLKQCFSLSD
`
`760
`761
`755
`371
`
`*
`Fig. 4. Amino acid sequence alignment of eukaryotic Seprase C-terminal. Alignment of amino acid sequences for the C-terminal of Seprase; deduced sequences Homo
`sapiens (AAC51668), Mus musculus (AAH19190), Xenopus laevis (AAC59872) and the putative sequence Bos Taurus (XP_603457). Alignment was performed
`using MultAlin [114] and edited using GeneDoc [115]. Sequence similarity is represented by grey scaling, with black being the highest similarity. The amino acids of
`the catalytic triad (Ser624, Asp702, His734) are indicated (⁎). The serine protease consensus motif G-X-S-X-G is underlined.
`
`Therefore, it may be concluded from this that Seprase activity is
`determined by the association of its subunits to form a proteo-
`lytically active dimer [27]. The proteolytic gelatinase activity of
`membrane-bound Seprase was found to be maximal at neutral
`pH and was enhanced by a mixture of 2 mM EDTA and 2 mM
`DTT (which inhibits metal-dependent proteases and activates
`cysteine proteases respectively) [11]. However, a previous study
`of the soluble form of Seprase demonstrated that EDTA had no
`effect on the proteolytic dipeptidyl peptidase activity and,
`contrary to previous reports DTT had a detrimental effect, with
`5 mM DTT causing a 10% loss of activity [41].
`A more recent study using a generated soluble recombinant
`Seprase (r-Seprase, 160-kDa), lacking cytoplasmic and trans-
`membrane domains, found that in the presence of putative
`EDTA sensitive activators, r-Seprase was converted into 70-
`kDa to 50 kDa shortened forms of Seprase (s-Seprase) [42].
`These shortened forms of Seprase exhibited a 7-fold increase in
`gelatinase activity, whereas levels of DPP activity remained
`unchanged [42]. Data shows that proteolytic truncation of the
`
`NH2-terminal peptides of Seprase reduces steric hindrance for
`the gelatin substrate but not the dipeptidyl peptidase substrate,
`thereby increasing the gelatinolytic activity of Seprase [42].
`Table 1 summarises some of the biochemical properties of
`Seprase.
`
`4. Purification and activity detection of Seprase
`
`Seprase has been purified from cell membranes and shed
`vesicles of LOX human amelanotic melanoma cells [3,11]. It
`has also been purified from 9-day old chicken embryos [43].
`Size exclusion chromatography (S-200) and affinity chromato-
`graphy using wheat germ agglutinin (WGA)-agarose are two of
`the most widely used resins for Seprase purification [11,12].
`Immunoaffinity purification of Seprase has also been utilised,
`with the mAb F19 pre-coated onto Sepharose CL-4B beads
`[10]. Soluble forms and isoforms of these membrane proteases
`are beginning to be found in biological fluids [15,25]. Recently
`our group has published the purification scheme for the soluble
`
`Table 1
`Biochemical properties of Seprase
`
`Source
`
`MW (kDa)
`
`pI
`
`pH
`optimum
`
`Temperature
`optimum (°C)
`
`Amino acid identity
`to human Seprase (%)
`
`GenBank
`accession number
`
`Reference
`
`5.0
`
`–
`5.68
`–
`
`7.0
`
`8.5
`7.5
`7.6
`
`–
`
`–
`–
`37
`
`100%
`
`100%
`–
`–
`
`AAC51668
`
`[3,11,12]
`
`–
`–
`–
`
`AAH19190
`AAC59872
`
`[40]
`[25,41]
`[43]
`
`[34]
`[33]
`
`Homo sapiens
`Malignant melanoma cells; LOX cells
`
`Recombinant; cDNA from WI38 cells
`Bovine serum
`Chicken embryo
`
`170 — dimer
`97 — subunit
`95 — subunit
`96 — subunit
`160 — dimer
`97 — subunit
`–
`–
`
`89%
`Mus musculus
`50%
`Xenopus laevis
`Summary of the published biochemical properties of Seprase. Those marked with (–) have not been published.
`
`–
`–
`
`–
`–
`
`–
`–
`
`Petitioner GE Healthcare – Ex. 1025, p. 1134
`
`

`

`P. O'Brien, B.F. O'Connor / Biochimica et Biophysica Acta 1784 (2008) 1130–1145
`
`1135
`
`form of Seprase from bovine serum [25]. This purification
`procedure involved a combination of hydrophobic interaction
`chromatography, hydroxylapatite, and cibacron blue chromato-
`graphy followed by size exclusion chromatography.
`Until recently, the most sensitive assay available for Seprase
`detection involved gelatin zymography, which exploits the
`established gelatinase activity associated with Seprase [12].
`However, this is not a quantitative assay. A semi-quantitative
`assay was developed based on the degradation of radiolabelled
`gelatin substrate and subsequent qualitative measurement of the
`released fragments [43]. Seprase was reported to possess prolyl
`dipeptidyl peptidase activity [15,32,40,44] and Ala-Pro-AFC was
`seen as a potential sensitive fluorogenic substrate. Interestingly,
`conflicting results exist with reports that Seprase has no
`such prolyl dipeptidyl peptidase cleavage activity [12,45].
`
`5. Tissue distribution of Seprase
`
`Studies have shown that Seprase is transiently expressed
`in certain normal fetal mesenchymal tissues, during wound
`healing and in reactive stroma responding to epithelial cancers
`and some sarcomas [7,8]. Normal adult
`tissues as well as
`malignant epithelial, neural and haematopoietic cells are
`generally Seprase-negative. The initial identification of Seprase
`involved the study of six surface glycoproteins that were
`differentially expressed during normal development, prolifera-
`tive activation and malignant transformation of mesenchymal
`cells and tissues [46]. The monoclonal antibody F19 was used
`to define the human cell surface glycoprotein Seprase. The F19
`antigen (now known as Seprase) was found to be expressed on
`cultured fibroblasts derived from various organs, several fetal
`mesenchymal tissues, scar tissue and a proportion of sarcoma
`cell lines. In normal adult tissues expression of the F19 antigen
`was restricted to occasional fibroblasts and to a set of pancreatic
`islet cells. The pattern observed in this initial study suggests that
`Seprase is a cell surface marker for proliferating mesenchymal
`cells and that its expression may be induced by normal growth
`factors or during malignant transformation [46].
`Another early study describes the induction of F19 in the
`reactive mesenchyme of epithelial tumours (carcinomas) [7].
`Fibroblasts positive for the F19 antigen (Seprase) using immuno-
`histochemical studies were found in primary and metastatic
`carcinomas including colorectal, breast, ovarian, bladder, and
`lung carcinomas. This study also analysed dermal
`incision
`wounds and found that F19 was strongly induced during scar
`formation. These studies suggest that the F19+ phenotype cor-
`relates with specialised fibroblast functions in wound healing,
`inflammation and malignant tumour growth [7]. Another im-
`portant observation from this study is that the cellular immuno-
`staining patterns obtained with the tumour tissue suggests that
`Seprase is localised exclusively in the cell membrane/cytoplasm
`of fibroblastic cells. This cellular staining is consistent with the
`cell surface localisation of Seprase in cultured fibroblasts [7].
`Since these early reports, more studies have shown that
`Seprase is expressed in reactive human stromal fibroblasts [23].
`Studies have confirmed the expression of Seprase in primary
`breast infiltrating ductal carcinoma, colon adenocarcinoma and
`
`lung adenocarcinoma and also in metastatic colon adenocarci-
`noma in the hepatic system [23,47]. Several groups have shown
`Seprase to be expressed in the reactive stromal fibroblasts of
`human breast cancer and its absence in normal breast tissue
`[7,10,16,48]. As mentioned above, Seprase is expressed by
`infiltrating ductal carcinoma (IDC) cells in breast cancer
`patients; however it is not expressed by normal breast epithelia
`[23,47,49]. Further work has shown that Seprase expression is
`not confined to stromal fibroblasts but that the protease is also
`expressed in some types of malignant cells of epithelial origin
`[50–52]. Stromal expression of Seprase in IDC of the breast was
`associated with longer survival of patients [4,15,48]. Contrary to
`this, a recent study of patients with epithelial ovarian carcinoma
`has shown that the overall survival decreased with expression
`of Seprase protein (p = 0.03) [52]. This report suggests that the
`conflicting data may result from the previous studies having
`either small samples and series or short follow-up period [52].
`Reports differ in the cellular localisation of FAPα and Seprase
`depicted by immunochemistry [15,49]. The apparent difference
`is thought to be partially due to the use of antibodies that
`recognise, with varying affinity, different epitopes exhibited by
`FAPα (derived from activated fibroblasts) and Seprase (derived
`from invasive cancer cells).
`Expression patterns of Seprase were examined in cervical
`carcinoma and cervical intraepithelial neoplasm [50]. This em-
`braces both carcinoma in situ and the precursor lesions known as
`dysplasia or ‘disordered differentiation’. Some micro-invasive
`carcinomas and all
`invasive carcinomas showed Seprase
`immunoreactivity in the cancer cells [50]. The findings in this
`study show a direct correlation between gelatinase expression
`and the malignant phenotype. Thus Seprase may be an early
`marker of tumour progression characterising Seprase expression
`with invasive growth. A separate study to support the concept
`was performed by Iwasa et al. [4], who examined Seprase
`expression in colorectal cancer specimens. Immunoblotting
`showed higher levels of Seprase protein in the cancer tissue than
`in normal colorectal tissue (p b 0.001). The results also revealed
`a significant correlation between Seprase expression and lymph
`node metastasis (p = 0.033).
`Expression patterns of Seprase in human gastric cancer were
`investigated using immunohistochemistry and the study showed
`that there were distinct differences in its expression between
`intestinal- and diffuse-type gastric cancer [51]. Results also
`showed, as in Iwasa et al. [4], a correlation between Seprase
`expression and depth of invasion. In intestinal cancer, the stromal
`expression of Seprase significantly correlated with liver metastasis
`(p = 0.0002) and lymph node metastasis (p b 0.0001). In contrast,
`in diffuse-type cancer there was no correlation between stromal
`Seprase expression and lymph node metastasis (p = 0.0821) [51].
`A separate study looked at the expression of Seprase at the mRNA
`and protein level. This study found that Seprase-expressing
`carcinoma tissues were more prominently found in the scirrhous
`type than in other types of gastric carcinoma [53].
`Immunohistochemical studies have shown Seprase expres-
`sion was induced in patients with idiopathic pulmonary fibrosis
`(IPF) [54]. Its expression pattern is restricted to fibroblasts in
`areas of ongoing tissue injury (Fig. 3 of Ref. [54]). Seprase was
`
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`recently identified for the first time on chondrocyte membranes
`under conditions that promoted cartilage resorption and ele-
`vated expression in cartilage from osteoarthritis (OA) patients
`[55]. The results from this study supported a role for Seprase in
`the mechanisms leading to cartilage degeneration in OA. Gene
`expression profiling in the murine model showed a 7-fold
`increase in Seprase expression in inflamed, compared to non-
`inflamed paws [56].
`A study by Huber et al. [57] found that Seprase was
`expressed in benign and malignant melanocytic skin tumours.
`This is in contrast to the findings in benign epithelial tumours, in
`which little or no expression of Seprase was observed on stromal
`fibroblasts [7,47]. Normal adult skin, however, did not have any
`detectable Seprase activity. These contradictory results could be
`explained if melanocytic naevi (moles) are considered as
`precursor lesions for melanoma development, characterised by
`constitutively-active tumour stroma [57,58]. Gene expression
`studies have identified Seprase to be uniquely overexpressed in
`aggressive fibromatosis [59]. Aggressive fibromatosis is locally
`invasive but rarely metastasises. There are histologic similarities
`between this disease and the proliferative phase of wound healing.
`To further support the concept that Seprase is expr