THE EFFECT OF THE PROTEIN MATRIX PROXIMITY ON GLYCAN REACTIVITY IN A GLYCOPROTEIN MODEL

A series of biotinylated glycan-Asn derivatives has been synthesized c ontaining either no extension arm between biotin and Asn (glycan-bioti nyl Asn) or containing HN(CH2)(n)CO extension arms of differing length s, where n denotes the number of methylene groups in the arm (glycan-b iotinyl[HN(CH2)(n)CO]Asn, n = 1-5). The glycan structures used were Ma n(6)GlcNAc(2)-, Man(5)GlcNAc(2)-, GlcNAcMan(5)GlcNAc(2)- and Gal(2)Glc NAc(2)Man(3)GlcNAc(2)-, the substrates for mannosidase I, GlcNAc trans ferase I, mannosidase II and sialyltransferase, respectively. Each fam ily 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 pr oximity of the protein matrix on the glycan substrate quality. Mannosi dase 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 su bstrate. In contrast, GlcNAc transferase I and mannosidase II, which w ere both strongly inhibited by the proximal substrate complexes (no ex tension arm) showed complete release of the inhibition even with the s hortest (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 dist al complexes. The results show that the availability of different glyc an substrates in a given protein environment reflects, to a great exte nt, the nature of each individual enzyme. The mechanisms by which the protein matrix affects glycan processing are proposed to involve simpl e steric effects, as well as more subtle effects of the protein in per mitting or preventing certain active glycan conformations to form.