A New Type of Congenital Disorders of Glycosylation (CDG-Ii) Provides New Insights into the Early Steps of Dolichol-linked Oligosaccharide Biosynthesis

Abstract

Deficiency of GDP-Man:Man 1 GlcNAc 2 -PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man 1 GlcNAc 2 -PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man 1 GlcNAc 2 -PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an ␣1,3-mannosyltransferase. The resulting Man␣1,3-ManGlcNAc 2 -PP dolichol is further elongated by a yet unknown ␣1,6-mannosyltransferase.Congenital disorders of glycosylation (CDG) 1 compose a rapidly growing group of inherited multisystemic disorders in man, which are commonly associated with severe psychomotor and mental retardation (1). The characteristic biochemical feature of CDG is defective glycosylation of proteins due to mutations in genes required for the biosynthesis of N-linked oligosaccharides.The attachment of oligosaccharide chains onto newly synthesized proteins is one of the most widespread forms of co-and post-translational modifications and is found in animals, plants, and bacteria. Glycoproteins are located inside cells predominantly in subcellular organelles and in cellular membranes and most abundantly in extracellular fluids and matrices. The oligosaccharide moiety of the glycoproteins can affect their folding, their transport, as well as their biological activity and stability (2, 3). The complex process of protein glycosylation requires more than a hundred glycosyltransferases, glycosidases, and transport proteins. CDG are classified into two groups. Defects of the assembly of lipid-linked oligosaccharides or their transfer onto nascent glycoproteins compose CDG type I, whereas CDG type II includes all defects of trimming and elongation of N-linked oligosaccharides (4). In the past 7 years the molecular nature of eight CDG-I and four CDG-II types could be identified (5-24).Here we describe for the first time a molecular defect in glycoprotein biosynthesis in man which affects at the cytosolic side of the endoplasmic reticulum the transfer of mannosyl residues from GDP-Man to Man 1 Glc...

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

“…Together with the experimentally determined enzyme activity of Alg2 here, one can now postulate that the temperature-sensitive alg2 yeast mutant allows a small extent mannose addition to Man 1 GlcNAc 2 -PP-Dol, because of some residual activity, and thus also results in accumulation of Man 2 GlcNAc 2 -PPDol, which is not the case in the null mutant of R. pussilis. In this argumentation also, results seamlessly incorporate, deriving from the analysis of the enzyme activity from human skin fibroblasts of a patient with a mutation in hAlg2 causing a defect in elongating Man 1 GlcNAc 2 -PP-Dol (21). In this study it was also shown that Man(1,6)ManGlcNAc 2 -PP-Dol tetrasaccharide did not act as acceptor in the patient but was elongated when membranes from control fibroblasts were used as enzyme source, albeit less efficiently than with Man 1 GlcNAc 2 -PP-Dol as acceptor (21).…”

“…In this argumentation also, results seamlessly incorporate, deriving from the analysis of the enzyme activity from human skin fibroblasts of a patient with a mutation in hAlg2 causing a defect in elongating Man 1 GlcNAc 2 -PP-Dol (21). In this study it was also shown that Man(1,6)ManGlcNAc 2 -PP-Dol tetrasaccharide did not act as acceptor in the patient but was elongated when membranes from control fibroblasts were used as enzyme source, albeit less efficiently than with Man 1 GlcNAc 2 -PP-Dol as acceptor (21). This latter observation holds also true for the Alg2 transferase from yeast (supplemental Fig.…”

“…Labeled glycolipids were then extracted and washed, and oligosaccharides were released from the dolichol-PP moiety by mild acid and analyzed by HPLC as described (27). Man[ 14 C]GlcNAc 2 -PP-Dol was prepared as described (21). For assay II, determination of enzyme activity by immunoprecipitation of Alg2-ZZ was carried out as follows.…”

“…8). On the other hand, biochemical studies in human fibroblasts from a patient with a defect in the human ALG2 ortholog, causing congenital disorder of glycosylation type CDG1i, pointed to a role in the transfer of the second, ␣1,3-linked mannose residue, because no elongation of Man(1,6)ManGlcNAc 2 -PP-Dol occurred (21). In contrast, control fibroblasts were able to do so, albeit with reduced efficiency when compared with Man(1,3)ManGlcNAc 2 -PP-Dol as glycosyl acceptor.…”

A regulatory SNP in AKAP13 is associated with blood pressure in Koreans

“…The SNP located on the upstream of asparagines-linked glycosylation 2 alpha-1,3-mannosyltransferase homolog (ALG2). 29 The ALG2 gene has three alternative transcripts, and the SNP locates at À3522 bp upstream of all three transcripts. The minor allele of this SNP was predicted by the TF search program to create a new cAMP response element-binding (CREB) to the ALG2 promoter.…”