Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human .beta.-hexosaminidase

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Abstract
Human .beta.-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexasominidase A, with the structure .alpha.(.beta.a.beta.b), and hexasaminidase B, 2(.beta.a.beta.b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the .beta.b chain, and one non concanavalin A binding glycopeptide, localized to the .beta.a chain, were found associated with the .beta.-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the .alpha. subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the .alpha. and one of the .beta.b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the .beta.b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the .beta.a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the .alpha. and .beta. subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the .alpha. chain we found only one possible site for in vivo phosphorylation. In the .beta. it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an .alpha.1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the .beta.b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.

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