Specificity of Solubilized Yeast Glycosyl Transferases for Polyprenyl Derivatives

Abstract

Four glycosyl transferases solubilized by detergents from Saccharomyces cerevisiae membranes have been tested for their specificity towards various polyprenols differing in chain length and saturation.1. The formation of dolichyl diphosphate-(N-acetylglucosamine)l-2 from uridine diphosphate N-acetylglucosamine and dolichyl monophosphate showed an obligatory requirement for a-saturated polyprenols. The apparent affinity for a-saturated polyprenyl phosphates improves with increasing chain length; those shorter than seven isoprene units were inactive.2. The rate of transfer of the chitobiosyl unit from dolichyl diphosphate chitobiose to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val followed the sequence :3. The enzyme transferring the mannosyl group from guanosine diphosphate mannose to dolichyl monophosphate also showed a clear dependence on a-saturation and chain length. In this case the chain length affects mainly the maximal velocity of the reaction. The highest value was obtained with C55 a-dihydropolyprenyl phosphate (100 nmol x h-' x mg protein-'), whereas the value with C35 a-dihydropolyprenyl phosphate was only 8 nmol x h-' x mg protein-'.4. The mannosyl transfer from dolichyl monophosphate mannose to dinitrophenylated tetrapeptide Asn-Ala-Thr-Vol to form on O-glycosidic bond strongly depended on a-saturation and chain length of the polyprenyl phosphate. The rate of transfer correlated directly with the length of the polyprenyl residue. With c 8 0 a-dihydropolyprenol less than half the rate of the C~O O compound was observed, whereas C55 and C X a-dihydropolyprenol are only 20 and 10 % as active, respectively.The biosynthesis of asparagine-linked oligosaccharide units of glycoproteins most likely occurs in all eucaryotic cells by a rather complex sequence of reactions which has been called the dolichol pathway [l-51. An oligosaccharide consisting of 14 hexose units is first built up sequentially onto dolichyl phosphate followed by a transfer en bloc of the preformed oligosaccharide to the nascent polypeptide chain. Subsequently part of the sugar residues is removed and thereafter sugars which compose the outer branches of complex type oligosaccharides are added [6 -81. In addition dolichyl phosphate has been shown to be involved also in the formation of the O-glycosidic linkage of glycoproteins ; however, up to now this role has only been demonstrated for fungal cells [5].

The biological role of dolichol

“…a-Saturation of dolichyl phosphate is a requirement for its reaction with activated nucleotidesugars [35][36][37]. The isoprene length of polyisoprenes occurring in animal cells has no influence on the construction of the sugar chain: all individual dolichols react equally well with the different activated monosaccharides [37][38][39]. The short dolichyl phosphate species are poor sugar acceptors, but since they are absent from animal cells, this finding has no physiological importance.…”

“…Information about the importance of polyisoprenoid structure to the glycosylation process is also available (Table 5). a-Saturation of dolichyl phosphate is a requirement for its reaction with activated nucleotidesugars [35][36][37]. The isoprene length of polyisoprenes occurring in animal cells has no influence on the construction of the sugar chain: all individual dolichols react equally well with the different activated monosaccharides [37][38][39].…”

Structure-Function Analysis of the Dolichyl Phosphate-Mannose: Protein O-Mannosyltransferase ScPmt1p

“…ScPmt1p-catalyzed transfer of mannose from Dol-P-␤-D-mannose (with inversion of its anomeric configuration) to serine/threonine residues of specific protein acceptors can be assayed both in vitro and in vivo (18,(22)(23)(24). Thereby, a strict stereospecificity for Dol-P-␤-D-mannose and the recognition of the ␣-isoprene unit of the dolichyl moiety has been observed (25,26). In vitro studies have shown that ScPmt1p glycosylates a restricted spectrum of peptides (18,27,28).…”

Identification and characterization of a cis,trans‐mixed heptaprenyl diphosphate synthase from Arabidopsis thaliana

“…Dolichol rather than polyprenol is considered to be utilized as the glycosyl carrier lipid, especially in seed plants [6]. Moreover, it has been shown that the substrate specificities of glycosyl transferases for Z,E-mixed polyisoprenoids in yeast depend not only on the a-saturation but also on the chain length, and polyisoprenoids shorter than C 55 are hardly utilized as a glycosyl carrier lipid in vitro [16]. Therefore, in trees, the physiological role of the shortchain betulaprenols is considered to be the provision of mechanical resistance against frost-caused breaks in cold climates [5].…”