Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H

Abstract

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.