Transfer of N,N′-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Abstract

Gray matter and white matter membranes catalyze the transfer of label from UDP- N-acetyl-[ 14C ] glucosamine into N-acetyl[ 14C ]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [ 14C ]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[ 14C ]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[ 14C ]glucosamine-labeled glycoprotein reveals two N-acetyl[ 14C ]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β- N-acetylglucosaminidase. A smaller N-acetyl[ 14C ]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N, N′-diacetyl[ 14C ]chitobiose. The linkage between the 14C -labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[ 14C ]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[ 14C ]glucosamine-labeled glycoprotein after incubation with UDP- N-acetyl[ 14C ]glucosamine. When gray matter membranes are incubated with UDP- N-acetyl[ 14C ]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[ 14C ]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[ 14C ]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled 14C -labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[ 14C ]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the 14C -labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [ 14C ]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.