Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2.
The lysosomal membrane glycoproteins lamp-1 and lamp-2 are extensively glycosylated with a variety of different carbohydrate structures of both N-linked and O-linked type. In the present paper, we report the localization of O-linked oligosaccharides exclusively to the hinge-like regions of lamp-1 and lamp-2 isolated from human chronic myelogenous leukemia cells. In both glycoproteins, the O-glycans appear in clusters. In lamp-1, Thr-171, Thr-172, Ser-179, Ser-181, and Ser-183 were fully glycosylated, whereas Ser-169 was partially glycosylated. In lamp-2, complete glycosylation was found at Ser-167, Thr-168, Thr-172, Thr-175, Thr-176, Thr-182, and Thr-183, and partial glycosylation at Ser-179 and Thr-181, and possibly also at Thr-185. The amino acid sequences of these O-glycosylation sites are consistent with the previous reports that residues at positions -1 and +3 may influence the glycosylation reaction. Circular dichroism and nuclear magnetic resonance spectroscopy was used for the structural characterization of a synthetic peptide corresponding to residues 167 to 190 of lamp-1. The results indicated that the proline-rich O-glycan acceptor region does not adopt any typical periodic structure but differs from random-coil structure. The circular dichroism spectrum of the peptide is, however, similar to that of porcine submaxillary apomucin. A significant conformational variability was observed in this region, presumably due to a slow (on the nuclear magnetic resonance time scale) cis-trans isomerization of several proline residues. These results, taken together, strongly suggest that a hinge region does not display any typical ordered structure. The presence of O-glycans thus likely protects this region from intralumenal lysosomal proteases.