Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by (1-O)galactosyl-and ␣(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen (1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.Collagens are the most abundant proteins in the human body. To date, 29 types of collagen have been described, which are encoded by at least 44 genes (21, 37, 45). Collagens are characterized by domains representing repeats of the triplet Gly-X-Y, where proline and lysine are often found at positions X and Y. The Gly-X-Y repeats are not confined to collagens but are also found in several other proteins, such as the hormone adiponectin (29), the mannose-binding lectin (MBL) (11), the C1q complement protein (35), the COLQ subunit of the acetylcholine esterase complex (4), and the surfactant proteins SP-A and SP-D (11).After synthesis in the endoplasmic reticulum (ER), three procollagen subunits associate to build a right-handed triple helix. However, before the formation of the triple-helix structure, the nascent procollagen polypeptides undergo several posttranslational modifications. These modifications comprise the hydroxylation of selected proline (20) and lysine (33) residues, which are catalyzed by three prolyl-4-hydroxylases (17), one prolyl-3-hydroxylase (46), and three lysyl hydroxylases (43). Hydroxylysine can be further modified by the addition of the monosaccharide Gal(1-O) or the disaccharide Glc(␣1-2)Gal(1-O) (39).Whereas the glycosylation of collagen was first described by Grassmann and Schleich in 1935 (9) and the structure of the glycan determined by Spiro in 1967 as being Glc(␣1-2)Gal(1-O)Hyl (40), the molecular nature of the collagen glycosyltransferase enzymes has remained elusive up to now. Collagen galac...
Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipidlinked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98 -100% occupancy for all Nglycosylation sites of transferrin and ␣ 1 -antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in ␣ 1
Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.