Identification and functional analysis of a defect in the human ALG9 gene: definition of congenital disorder of glycosylation type IL

Abstract

Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG). Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects. Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha 1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc(2)Man(8) structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein. A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene. The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene. The ALG9 defect found in the patient with CDG--who presented with developmental delay, hypotonia, seizures, and hepatomegaly--shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology. The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."

Figure 1

[³H]Mannose-labeled lipid- and N-linked oligosaccharide profiles from human primary fibroblasts. A, Separation of LLOs from normal control fibroblasts. B, Separation of LLOs from patient fibroblasts. C, Separation of NLOs from patient fibroblasts. Arrows mark the elution times of standard oligosaccharides from yeast (M5–M9, GlcNAc₂Man_5–9; G1–G3, GlcNAc₂Man₉Glc_1–3).

Figure 2

LLO biosynthesis pathway in the ER lumen. Arrows mark the reactions in which dolichol-phosphate (DolP)–Man or DolP-Glc are used as substrates and where the DolP-Man–dependent transferases ALG3, ALG9, and ALG12 are involved. The complete oligosaccharide GlcNAc₂Man₉Glc₃ is transferred from its lipid carrier to nascent polypeptides by the oligosaccharyltransferase complex (OST). Known types of CDG are marked with the respective names CDG-Ic, -Id, -Ig, and -Ih.

Figure 3

ALG9 protein sequence comparison. Amino acid sequences of ALG9 proteins from Homo sapiens (Hs) (Entrez accession number AAL25798), Caenorhabditis elegans (Ce) (Entrez accession number CAA90107), Drosophila melanogaster (Dm) (Entrez accession number NP_651353), Schizosaccharomyces pombe (Sp) (Entrez accession number CAB75773), and S. cerevisiae (Sc) (Entrez accession number CAA96122) were aligned using the ClustalW program (Thompson et al. 1994). The amino acid change E523K is indicated for the human ALG9 protein.

Figure 4

Complementation of Δalg9 wbp1-2 yeasts with human ALG9 cDNAs. Transformants were spotted in sixfold dilutions on YPD plates and were incubated at 23°C, at 30°C, or at 33°C for 120 h. Transformants carried either the vector alone (“mock”), a plasmid complementing the wbp1-2 mutation (“WBP1”), the pALG9 plasmid expressing the S. cerevisiae ALG9 gene (“ScALG9”), or plasmids expressing the normal human ALG9 cDNA or the CDG patient ALG9[E523K] cDNA.