The effect of the protein matrix proximity on glycan reactivity in a glycoprotein model

A series of biotinylated glycan-Asn derivatives has been synthesized containing either no extension arm between biotin and Asn (glycan-biotinyl Asn) or containing HN(CH2)nCO extension arms of differing lengths, where n denotes the number of methylene groups in the arm (glycan-biotinyl[HN(CH2)nCO]Asn, n = 1-5). The glycan structures used were Man6GlcNAc2-, Man5GlcNAc2-, GlcNAcMan5GlcNAc2- and Gal2GlcNAc2Man3GlcNAc2-, the substrates for mannosidase I, GlcNAc transferase I, mannosidase II and sialyltransferase, respectively. Each family of substrates was subjected to the action of its respective enzyme in the absence and in the presence of streptavidin, and the relative rate of processing (in the presence of UDP-GlcNAc and the mannosidase II inhibitor, swainsonine for GlcNAc transferase I and CMP-sialic acid for sialyl transferase) was measured to evaluate the effect of the proximity of the protein matrix on the glycan substrate quality. Mannosidase I was found to be strongly inhibited by the protein matrix in the proximal as well as in the distal positions relative to the glycan substrate. In contrast, GlcNAc transferase I and mannosidase II, which were both strongly inhibited by the proximal substrate complexes (no extension arm) showed complete release of the inhibition even with the shortest (n = 1) extension arm. Sialyl transferase showed inhibition of both reaction steps in the proximal complex, and complete release of the inhibition of the first step, but not the second step, in the distal complexes. The results show that the availability of different glycan substrates in a given protein environment reflects, to a great extent, the nature of each individual enzyme. The mechanisms by which the protein matrix affects glycan processing are proposed to involve simple steric effects, as well as more subtle effects of the protein in permitting or preventing certain active glycan conformations to form.