ALG6-CDG

What is ALG6?

ALG6 is the name of a gene that encodes an enzyme called alpha-1,3-glucosyltransferase. That’s actually an abbreviation. It’s full name is the intimidating “dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase.” Let’s stick with the moniker ALG6. 

ALG6 protein is threaded into the endoplasmic reticulum (ER) membrane. Its business end is in the ER lumen. Its enzymatic function is to transfer the first glucose building block to a maturing lipid-linked oligomannose core. The oligosaccharide product of ALG6 is the substrate for ALG8, the glucosyltransferase that adds a second glucose. The oligosaccharide product of ALG8 is the substrate for ALG10, the glucosyltransferase that adds a third glucose, yielding the 14-sugar oligosaccharide called Glc3Man9GlcNAc2
Dolichol-linked Glc3Man9GlcNAc2 is the substrate of the oligosaccharyltransferase (OST) complex, which transfers the oligosaccharide to an asparagine side chain of a maturing glycoprotein.

Orthologs of ALG6 are conserved across mice, zebrafish, flies, worms, budding yeast and fission yeast.

What does ALG6 do?

Yeast geneticists in the 1980s and 1990s created the gene prefix ALG, which stands for ALtered in Glycosylation. ALG is the yeast cell version of CDG. Like many CDG genes, the function of ALG6 was first studied in budding yeast (Runge et al., 1984). A rich yeast literature strived for two decades and then the use of yeast models of CDGs practically disappeared from the literature starting at the turn of the millennium. 

It’s a shame because the basic biology of ALG6 and the assembly line of glycosyltransferases and mannosyltransferases was worked out in yeast over two decades ago: “In the yeast Saccharomyces cerevisiae, alg mutations which affect the assembly of the lipid-linked oligosaccharide at the membrane of the endoplasmic reticulum result in the accumulation of lipid-linked oligosaccharide intermediates and a hypoglycosylation of proteins.” (Reiss et al., 1996).

The foundation of ALG6-CDG yeast modeling research is Imbach et al., 1999. ALG6 is essential for yeast viability so knocking it out results in lethality. Expression of human ALG6 in a yeast mutant lacking yeast ALG6 rescued lethality, and partially rescued the glycosylation defect of a secreted protein called mCPY (the yeast analog of serum transferrin).

Figure 2B from Imbach et al., 1999 showing in bold the amino acids that have remained unchanged in comparison between yeast ALG6 and human ALG6 despite over a billion years of separate evolution.
Figure 5 from Imbach et al., 1999. On the left, the full glycosylation status of mCPY is partially restored by expression of human ALG6, but expression of human ALG6 with a damaging mutation A333V does not rescue. On the right, yeast cell growth was rescued by expression of human ALG6, but not by expression of human ALG6 with a damaging mutation A333V.

Clinical presentation of ALG6-CDG

ALG6-CDG (formerly known as CDG-1c) is the second most common CDG after PMM2-CDG. Like PMM2-CDG, ALG6-CDG has a variable multisystem clinical presentation expect milder and the primary organ system affected is the brain. First clinical report is Imbach et al., 2000. Unfortunately there aren’t many open access clinical reports.

Broader implications beyond ALG6-CDG

The F304S mutation in ALG6 is present in about 1 in 3 people. Turns out the presence of F304S in ALG6 can exacerbate PMM2-CDG disease progression. PMM2-CDG patients with a more severe disease progression were more likely to carry the F304S mutation in ALG6 (Westphal et al., 2002). The variable multisystem clinical presentation of CDGs and the notorious lack of genotype-phenotype relationships when comparing CDG gene mutations to disease severity may be two sides of the same coin. And the street goes both ways. If a mutation in a CDG gene can exacerbate PMM2-CDG, then there are CDG gene mutations out there that have a protective effect. The network of disease modifier relationships among CDG genes needs to be mapped.

Next steps toward the clinic

The template for a budding yeast model is Imbach et al., 1999. However, reviving ALG6-CDG yeast models will require using codon-optimized human ALG6 and a range of protomer strengths in rescue experiments, as described in Lao et al., 2019

Fission yeast model is ready and raring to go, just need some researchers to step up (Fanchiotti et al., 1998). Worm, fly, zebrafish and mouse models are all lacking. Model organism researchers, please rise to the challenge. ALG6-CDG patient fibroblasts have already been validated. From there drug repurposing screens offer the fastest and cheapest path to the first approved treatment for ALG6-CDG. Identify a bridge therapy that buys time for gene therapy and gene editing technologies to mature. In the case of ALG6 splice mutations, personalized antisense oligonucleotides (ASOs) offer hope.

References

Fanchiotti S, Fernández F, D’Alessio C, Parodi AJ. (1998). The UDP-Glc:Glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress. Journal of Cell Biology. 143: 625-635.

Imbach T, Burda P, Kuhnert P, Wevers RA, Aebi M, Berger EG, Hennet T. (1999). A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic. Proc Natl Acad Sci U S A. 96: 6982-7.

Imbach T, Grünewald S, Schenk B, Burda P, Schollen E, Wevers RA, Jaeken J, de Klerk JB, Berger EG, Matthijs G, Aebi M, Hennet T. (2000).Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic. Human Genetics. 106: 538-545.

Lao JP, DiPrimio N, Prangley M, Sam FS, Mast JD, Perlstein EO. (2019). Yeast Models of Phosphomannomutase 2 Deficiency, a Congenital Disorder of Glycosylation. G3. 9: 413-423.

Reiss G, te Heesen S, Zimmerman J, Robbins PW, Aebi M. (1996). Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway. Glycobiology. 6: 493-498.

Runge KW, Huffaker TC, Robbins PW. (1984). Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway. Journal of Biological Chemistry. 259: 412-417.

Westphal V, Kjaergaard S, Schollen E, Martens K, Grunewald S, Schwartz M, Matthijs G, Freeze HH. (2002). A frequent mild mutation in ALG6 may exacerbate the clinical severity of patients with congenital disorder of glycosylation Ia (CDG-Ia) caused by phosphomannomutase deficiency. Human Molecular Genetics. 11: 599-604.

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