Differential accessibility of the carbohydrate moieties of C.hivin.1s-C.hivin.1r-C.hivin.1r-C.hivin.1s, the catalytic subunit of human C.hivin.1

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Abstract
The catalytic subunit of human C.hivin.1, C.hivin.1s.sbd.C.hivin.1r.sbd.C.hivin.1r.sbd.C.hivin.1s, is a Ca2+-dependent tetrameric association of two serine proteases, C.hivin.1r and C.hivin.1s, which are glycoproteins containing asparagine-linked carbohydrates. With a view to investigate the accessibility and the possible functional role of these carbohydrates, the isolated proteases and their Ca2+-dependent complexes were submitted to deglycosylation by peptide:N-glycosidase F, an endoglycosidase that specifically hydrolyzes all classes of N-linked glycans. Treatment of isolated C.hivin.1r and C.hivin.1s led to the removal of the carbohydrate moieties attached to their N-terminal .alpha. region, whereas those located in the C-terminal .gamma.-B catalytic domains were resistant to hydrolysis. Formation of the Ca2+-dependent C.hivin.1s.sbd.C.hivin.1s dimer and C.hivin.1s.sbd.C.hivin.1r.sbd.C.hivin.1r.sbd.C.hivin.1s tetramer induced specific protection of the single carbohydrate attached to the .alpha. region of C.hivin.1s and of one of the two carbohydrates located in the corresponding region of C.hivin.1r. Sequence studies indicated that the carbohydrates protected upon homologous (C.hivin.1s.sbd.C.hivin.1s) or heterologous (C.hivin.1r.sbd.C.hivin.1s) interactions are attached to asparagine residues 159 of C.hivin.1s and 204 of C.hivin.1r, at the C-terminal end of the EGF-like domain of both proteases. These data bring further evidence that Ca2+-dependent interactions between C.hivin.1r and C.hivin.1s are mediated by their N-terminal .alpha. regions and strongly suggest that, inside these regions, the EGF-like domains play an essential role in these interactions.