Isolation and characterization of proteoglycans from human follicular fluid

Abstract

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0 x 106 Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (M(r) 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-α-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1.1 x 106 Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a 'composite' structure of this proteoglycan.