Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I

Abstract

This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I [MIM 605907]), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis. Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy. One patient had a homozygous point mutation (c.773C-->T, S258L), whereas the other patient was compound heterozygous for the mutations c.773C-->T and c.1025A-->C (E342P). Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles.

Figure 1

IEF of serum transferrin. The numbers on the right describe the numbers of sialic acids. Transferrin has two branched carbohydrate side chains, each bearing two sialic acids at the end. Fully glycosylated transferrin has four sialic acids; higher branching leads to increased numbers of sialic acids in a small fraction of the protein. Patients with CDG-Ia have either one or both carbohydrate side chains missing in about half of the transferrin population. The hypoglycosylation in CDG-Ik is much more severe.

Figure 2

TLC of dolichol-linked oligosaccharides. Fibroblasts were labeled with [³H]glucosamine for 1 h, and dolichol-linked oligosaccharides were extracted with chloroform:methanol (3:2), as described. Lanes 1–3 show fibroblasts (GM, NK = CDG-Ik fibroblasts; c = control fibroblasts). Lanes 4–5 show yeast fibroblasts (wt = wild type; PRY56 = ALG1 mutant). 1 = migration of higher–molecular-weight dolichol-linked oligosaccharides, 2 = migration of the dolichol-linked oligosaccharides accumulated in the ALG1 mutant.

Figure 3

In vitro elongation of GlcNAc₂-PP-dolichol (A) or Man₁GlcNAc₂-PP-dolichol (B). NK and GM are patients with CDG-Ik. [¹⁴C]-labeled GlcNAc₂-PP-dolichol was incubated in the presence of detergent-solubilized membrane extracts from control and patient fibroblasts. Dolichol-linked oligosaccharides were analyzed by HPLC. In control extracts, nearly half of the substrate A). To demonstrate that the subsequent mannosylation reaction was unaffected, Man₁[¹⁴C]GlcNAc₂-PP-dolichol was used as substrate (B). In both patients with CDG-Ik (B.c, B.d), elongation occurred equal to that in the control (B.a, B.b). GN = N-acetylglucosamine; M = mannose.

Figure 4

Growth characteristics of transformed PRY56 yeast cells showing 10-fold dilutions from top to bottom. Pictures were taken after 1 wk at the permissive temperature (26°C, left) or at the restrictive temperature (36°C, right). From left to right, wild-type transformed cells, transformed cells with the 1025A→C (NK) allele, transformed cells with the 773C→T allele (both patients), and mock-transformed cells.

Figure 5

Glycosylation of CPY. ALG1-deficient yeast (PRY56) is shown on the left; wild-type yeast (wt) is on the right. “c−” denotes ALG1 yeast transformed with the human wild-type homologue, without copper induction; “c+” indicates ALG1 yeast transformed with the human wild-type homologue, with copper induction. “mock” designates mock-transformed yeast cells, without insert. “773C→T” denotes the transformed cells with the allele carrying the c.773C→T mutation, induced with copper. “1025A→C” indicates the transformed cells with the allele carrying the c.1025A→C mutation, induced with copper.