title: 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 3.0x10(6) Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 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-alpha-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. 1x10(6) 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.
authors.affiliation: Y Forskningslaboratorium, Department of Obstetrics and Gynaecology, Faculty of Health Sciences, Aarhus University Hospital, Skejby Hospital, DK-8200 Aarhus N, Denmark.
authors.forename: G V
authors.initials: GV
authors.lastname: Eriksen
authors.forename: I
authors.initials: I
authors.lastname: Carlstedt
authors.forename: M
authors.initials: M
authors.lastname: Mörgelin
authors.forename: N
authors.initials: N
authors.lastname: Uldbjerg
authors.forename: A
authors.initials: A
authors.lastname: Malmström
mesh_terms: D000468:Alkalies; D000510:Alpha-Globulins / Q000032:analysis / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D002809:Chondroitin Sulfates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D002853:Chromatography, Liquid; D005260:Female; D015571:Follicular Fluid / Q000737:chemistry*; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006497:Heparan Sulfate / Q000032:analysis; D006801:Humans; D037181:Lectins, C-Type; D008854:Microscopy, Electron; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011509:Proteoglycans / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D013455:Sulfur / Q000032:analysis; D053675:Versicans
chemical_list: D000468:Alkalies; D000510:Alpha-Globulins; D000596:Amino Acids; D011508:Chondroitin Sulfate Proteoglycans; D002809:Chondroitin Sulfates; D002810:Chondroitinases and Chondroitin Lyases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D037181:Lectins, C-Type; D009844:Oligosaccharides; D011509:Proteoglycans; D013455:Sulfur; D053675:Versicans; C508322:VCAN protein, human; C071927:perlecan; C011877:inter-alpha-inhibitor