Processing Enzymes Involved in the Deglucosylation of N ‐Linked Oligosaccharides of Glycoproteins: Glucosidases I and II and Endomannosidase

Abstract

Subsequent to the discovery that dolichol-linked oligosaccharides are involved in the N-glycosylation of proteins in eukaryotic cells [ 11, it became apparent that the carbohydrate portion of these molecules contains peripheral residues of glucose linked to polymannose-di-N-acetylchitobiose [2]. Further structural investigations demonstrated that the glucose residues occur in a trisaccharide sequence [3] in which a-glycosidic bonds link the constituents to each other, as well as to the polymannose unit [4] as shown in Figure 1.It moreover became evident that these glucose constituents are essential for the cotranslational en bloc transfer of the oligosaccharide to asparagine in the Asn-XSer (Thr) constellations in the polypeptide chain [ 5 , 6, 71. Since glucose is generally not observed in mature N-linked oligosaccharides, their presence in newly glycosylated proteins prompted the suggestion that trimming enzymes which can remove these saccharides must be present as components of the intracellular processing apparatus [S]. Indeed, reports from a number of laboratories soon indicated that a-glucosidases which can cleave the glucose residues from the N-linked parent oligosaccharide occur in microsomal fractions from various sources [4, 9, 101. Subsequently it became apparent that two distinct processing a-glucosidases coexist in the endoplasmic reticulum (ER) which release the terminal a 1 1 2 and the two more internal wl43-linked glucose residues, respectively [ 1 I], and were accordingly designated as glucosidase I and glucosidase 11 (Figure 1). Concurrently or subsequently, ER and Golgi situated a-mannosidases trim the N-linked oligosaccharide to ManSGlcNAcI [12] which in turn is acted upon by a number of glycosyltransferases to yield the large variety of complex carbohydrate units which have been observed in eukaryotes. In some instances, however, mannose excision is limited and untrimmed or incompletely trimmed polymannose oligosaccharides can be found in the mature glycoprotein, as in the polymannose oligosaccharides of thyroglobulin or the hybrid units of ovalbumin. In any case, glucose removal is a preCarbohydrates in Chemistry and Biology

MPDU1 mutations underlie a novel human congenital disorder of glycosylation, designated type If

“…Analysis of NLO from cells that were grown in the presence of deoxynojirimycin as glycosidase inhibitor supported the assignment of these peaks and suggested that complete Glc 3 Man 9 GlcNAc 2 was transferred to protein in all patients' fibroblasts (data not shown). These different oligosaccharide species can be explained by the action of deglucosylating enzymes (38) and ER-localized mannosidases (39) as well as UDP-glucose-dependent glucosyltransferase (40) on the complete Glc 3 Man 9 GlcNAc 2 oligosaccharide transferred to protein. Interestingly, we observed a novel group of protein-bound oligosaccharides in patients L and S. A putative Man 5 GlcNAc 2 oligosaccharide represented the major peak, but signals comigrating with the Man 4 GlcNAc 2 and the Man 6 GlcNAc 2 were also present.…”

Role of N -linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation

“…Glucosidase I, an integral membrane enzyme with a luminally oriented catalytic domain, rapidly acts to specifically remove the terminal a1-2-linked residue of the triglucosyl sequence, which in almost all eukaryotes is essential for the effective action of the oligosaccharyltransferase. The release of the remaining glucose components is effected by glucosidase II, which in cleaving the Glc remove1-3Glc and Glc remove1-3Man linkages shows a dual specificity that has been attributed to two distinct active sites [21]. This glycosidase is a soluble luminal enzyme which is primarily located in the ER.…”

“…Enhanced proteolytic degradation has moreover been found to serve in the regulation of metabolic pathways which are prone to wide physiological fluctuations [11 -13] as well as in a number of pathological states [14], including cystic fibrosis [15], a 1 -antitrypsin deficiency [16] and congenital goiter [17], in which mutated proteins are produced; furthermore certain viruses evade cellular immune defenses by promoting degradation of host components [18]. Studies from a number of investigators have indicated that the processing intermediates of N-linked oligosaccharides produced by the a-glucosidases and a-mannosidases of the endoplasmic reticulum (ER) and pre-or cis-Golgi compartments [1-5, [19][20][21][22] provide specific retention or recycling signals. Thus, for the first time a biological rationale has been given to the now well delineated processing that the newly attached glucosylated polymannose oligosaccharides undergo on their way to becoming mature N-linked units, which is a strikingly different approach from the simple sequential step-by-step sugar attachment which prevails in the biosynthesis of O-linked oligosaccharides [23].…”