Abstract
Hyaluronic acid-binding region and trypsin-link protein were prepared from bovine nasal cartilage proteoglycan complex after trypsin digestion. Binary complexes were reformed between trypsin-link protein and hyaluronic acid-binding region or hyaluronate. Upon trypsin treatment of these complexes, two fragments deriving from trypsin-link protein were characterized. One of them, of 20 kDa, corresponds in fact to a 140-amino acid long fragment and bears the glycosylated site of trypsin-link protein; it appears to be involved in proteoglycan/link protein interaction. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link protein with hyaluronic acid. A structural model of bovine trypsin-like protein depicting two distinct domains involved in hyaluronate and proteoglycan subunit interactions is proposed.
Highlights
Hyaluronicacid-bindingregionand trypsin-link [15],indicating that thetwo binding sites are maintained in protein were prepared from bovine nascaalrtilagepro- TLP
Upon trypsin treatment of these complexest,wo frag- One of them, the 18-kDa fragment, constitutes the N-terminal ments deriving from trypsin-link protein were char- moiety of TLP and bears a glycosidic side chain; the other, acterized
The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link prowteitnh hyaluronic the 27.5-kDa fragment, is notglycosylated and consistsof the
Summary
From the Laboratory of Proteins (Unit6AssocGe Centre National de la Recherche Scientifique No 1188 alliee a 1 ’Znstitut National de la Sante et dela Recherche Medicale), University of Paris V, 45 rue des Saints-Peres, F 75270 Paris Cedex 06. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link prowteitnh hyaluronic the 27.5-kDa fragment, is notglycosylated and consistsof the. Separation Methods-Fractogel TSK HW 55 (F) gel filtrations were performed with 25 mM ammonium bicarbonate, 1 mM EDTA, digest of the proteoglycancomplex[12]: one of them, the pH 7.8, buffer as eluent (buffer A) on an analytical scale on a 30 X hyaluronic acidbinding-region (HABR), derives fromthe 0.7-cm column, flow rate 0.5 ml/min or at a preparative scale on a 60. The abbreviations used are: HA, hyaluronic acid; PGS, proteogly- in 62.5 mM Tris-HC1, 2% SDS, 10% glycerol, pH 6.8 buffer with or can subunits; LP, link proteins; HABR, hyaluronic acid-binding region; TLP, trypsin-link protein; GdnHC1, guanidine hydrochloride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CNBr, cyanogen bromide; FPLC, fast protein liquid chromatography.
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