Differential accessibility of the carbohydrate moieties of Cls-Clr-Clr-Cls, the catalytic subunit of human Cl.
The catalytic subunit of human Cl, Cls-Clr-Clr-Cls, is a Ca2+-dependent tetrameric association of two serine proteases, Clr and Cls, 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 Clr and Cls 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 Cls-Cls dimer and Cls-Clr-Clr-Cls tetramer induced specific protection of the single carbohydrate attached to the alpha region of Cls and of one of the two carbohydrates located in the corresponding region of Clr. Sequence studies indicated that the carbohydrates protected upon homologous (Cls-Cls) or heterologous (Clr-Cls) interactions are attached to asparagine residues 159 of Cls and 204 of Clr, at the C-terminal end of the EGF-like domain of both proteases. These data bring further evidence that Ca2+-dependent interactions between Clr and Cls 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.
Aude
C A
CA
Département de Recherches Fondamentales (Unité INSERM 238), Centre d'Etudes Nucléaires de Grenoble, France.
Lacroix
M B
MB
Arlaud
G J
GJ
Gagnon
J
J
Colomb
M G
MG
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000302:isolation & purification; D003172:Complement C1; D003488:Cyanogen Bromide; D006020:Glycopeptides / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases
D002241:Carbohydrates; D003172:Complement C1; D006020:Glycopeptides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D006026:Glycoside Hydrolases; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide