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PRG‐4/SZP N‐ and C‐terminal domains: cloning, expression and characterization
Author(s) -
Jones A.R.C.,
Hughes C.E.,
Wainwright S.D.,
Flannery C.R.,
Caterson B.
Publication year - 2004
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/j.0959-9673.2004.369at.x
Subject(s) - microbiology and biotechnology , vitronectin , proteoglycan , recombinant dna , affinity chromatography , cartilage , biology , chemistry , extracellular matrix , biochemistry , anatomy , gene , fibronectin , enzyme
Proteoglycan‐4 (PRG‐4), also known as superficial zone protein/proteoglycan (SZP), is an approximately 345‐kDa mucinous proteoglycan that has been detected in a variety of tissues including cartilage, tendon, bone, heart and liver (Ikegawa et al . 2000). In the synovial joint, PRG‐4 is specifically synthesized by chondrocytes located in the superficial zone of articular cartilage and by some surface‐lining cells of the synovium (Schumacher et al . 1994). Sequence analyses have shown that the N‐ and C‐terminal vitronectin‐like domains of PRG‐4 may impart interesting functions relevant to synovial joint metabolism (Merberg et al . 1993; Flannery et al . 1999). The objective of this study was to investigate these potential functions, facilitated by the production of PRG‐4 N‐ and C‐terminal domains as recombinant proteins. Methods cDNAs for the human N‐terminal (exons 2–5) and bovine C‐terminal (exons 7–12) domains of PRG‐4 were obtained by RT‐PCR and cloned into the expression vector pMT‐BiP for inducible, secreted expression in Drosophila S2 cells. Proteins were purified using FLAG‐M2 antibody affinity chromatography and visualized by SDS‐PAGE and Western blotting with PRG‐4‐specific antibodies. The heparin‐binding properties of recombinant proteins were investigated using heparin affinity chromatography. The interactions of recombinant PRG‐4 domains with human plasminogen activator‐inhibitor (PAI)‐1 and bovine type‐II collagen were assayed using standard ELISA techniques. Results Stable cell lines have been generated that express human N‐terminal and bovine C‐terminal PRG‐4 domains. In both cases, two proteins have been purified, possibly due to a splice mechanism by the expression system. N‐terminal sequence data and Western blotting indicate that the two species in each case could represent full‐length and truncated proteins. Analyses of the two PRG‐4 N‐terminal domain species have confirmed the presence of a predicted heparin‐binding domain and indicate that the molecule can bind to PAI‐1, with binding activity localized towards its two somatomedin B domains. The somatomedin B domain of vitronectin is known to bind PAI‐1 (Seiffert 1997). Analyses of the two PRG‐4 C‐terminal species have demonstrated self‐association under nonreducing conditions and binding to heparin and PAI‐1. Discussion The exact role of PRG‐4 in the synovial joint is yet to be elucidated. However, these results point towards the interaction of the N‐ and C‐terminal domains of PRG‐4 with structural molecules such as type‐II collagen and heparin, and functional molecules such as PAI‐1, a serpin that is involved in the fibrinolytic cascade and cell adhesion. These properties are in addition to the well‐documented boundary lubricating activity of the central mucinous region of PRG‐4.