What is ALG1?
ALG1 is the name of a gene that encodes an enzyme called beta-1,4-mannosyltransferase. ALG1 takes GDP-mannose and chitobiosyldiphosphodolichol and turns them into GDP and beta-1,4-D-mannosylchitobiosyldiphosphodolichol (also known as Man:GlcNAc2-PP-dolichol).
ALG1 protein sits atop the production funnel anchored in the endoplasmic reticulum (ER) membrane. As a glucosyltransferase, ALG1 add the first mannose building block to lengthening and ultimately branching glycan chain on nascent glycoproteins. The business end of ALG1 is located in cytoplasm, which is also true for the next two glucosyltransferases down the line, ALG2 and ALG11. Zooming out, downstream of ALG1 are 13 other ER-resident glucosyltransferases that sequentially add a mannose building block and then three terminal glucoses to each dolichol-linked glycan.
Orthologs of ALG1 are conserved in mice, zebrafish, flies, worms, budding yeast and fission yeast.
What does ALG1 do?
The ALG in ALG1 stands for ALtered in Glycosylation. Budding yeast were indispensable as a model system for understanding the function of all ALG genes, including ALG1. In the 1980s through the early 2000s, half a dozen papers characterizing ALG1 mutants and ALG1 function were published, starting with a genetic screen for mutants that are temperature sensitive for the ability to glycosylate proteins (Huffaker & Robbins, 1982). The same lab demonstrated that ALG1 is an essential gene in yeast, meaning ALG1 knockout mutants are inviable (Albright & Robbins, 1990). The first proof that human ALG1 can rescue a yeast alg1 knockout mutant was supplied by (Takahashi et al., 2000).
Remarkably but not unusual for CDGs, pathophysiological features of ALG1-CDG are conserved between human cells and yeast cells. What’s more the same or similar assays and protocols can be optimized for use in both model systems!
For example, serum transferrin is the most widely recognized and most used clinical diagnostic marker in suspected cases of congenital disorders of glycosylation. Serum transferrin originates in the liver where it is secreted into the bloodstream and binds up iron. Invertase is an enzyme that budding yeast cells secrete into their surroundings that breaks down complex sugars into digestible fungal foodstuff like glucose. Both transferrin and invertase need to be fully glycosylated to function properly. Their glycosylation status is an evolutionarily conserved biomarker.
Figure 4 from Huffaker & Robbins 1982 showing hypoglycosylation of invertase secreted by alg1-1 mutant yeast cells grown at the nonpermissive temperature 36˚C (b) but secretion of fully glycosylated invertase at the permissive temperature 26˚C (d). The alg1-1 mutation is temperature sensitive meaning the ALG1 protein is stable and functional at 26˚C but is unstable and nonfunctional at 36˚C. The smear in lane b is equivalent to the discrete disialo and asialo hypoglycosylated forms of serum transferrin.
Clinical presentation of ALG1-CDG
ALG1-CDG is the third or fourth most common CDG (formerly known as CDG-1k). At least 60 ALG1-CDG patients have been reported in the literature. Like PMM2-CDG, ALG1-CDG has a variable multisystem clinical presentation expect milder and the primary organ system affected is the brain. Diagnostic criteria include developmental delay, hypotonia, seizures, microcephaly and intellectual disability.
In 2004, three clinical reports combined ALG1-CDG patient fibroblasts and yeast to characterize ALG1-CDG patient mutations. Grubenmann et al., 2004 developed a more sensitive assay to detect short lipid-linked oligosaccharides that contain only a few or no mannose building blocks. Using this more sensitive assay they definitively diagnosed a patient with ALG1-CDG with what looked like three pathogenic variants: S150R and D429E inherited from the mother, and S258L inherited from the father. Then they expressed these three variants in yeast and found that D429E was in fact a benign mutation whereas the other two variants were indeed pathogenic. Schwarz el al., 2004 studied the S258L mutation and showed accumulation of oligosaccharide precursors when using radiolabeled glucosamine versus radiolabeled mannose building blocks. Kranz et al., 2004 characterized a new mutation E342P.
Ng et al., 2016 describes the largest clinical cohort to date. It also is the first example of using complementary yeast CDG models to verify that ALG1 mutations revealed by clinical whole exome sequencing are actually pathogenic. Yeast ALG1-CDG disease models can rapidly and cheaply screen dozens of ALG1 variants – and that was 3-4 years ago.
Broader implications beyond ALG1-CDG
Valderrama-Rincon et al., 2012 discovered that a minimal set of four genes including ALG1 comprise a module that can endow normally protein-glycosylation-free bacteria with the baby steps of N-linked glycosylation. While an interesting proof of concept, the logical next technological leap is cell-free production of synthetic lipid-linked oligosaccharides as building blocks for cell-free protein production and engineering applications. Rexer et al., 2018 prototype the idea in a test tube with proof of concept results.
Next steps toward the clinic
CRISPR-knockouts of human cells are now possible but that doesn’t mean the ALG1-CDG community can ignore the proven value of yeast models. Especially when we consider that the ALG1 yeast literature goes back three decades. ALG1-CDG yeast models and ALG1-CDG1 patient cell models have gone hand in hand for genetic diagnosis, drug repurposing, drug discovery and biomarker discovery.
In the case of ALG1, worm, fly, zebrafish and mouse models are absent. Those animal model gaps must be addressed before ALG1-CDG research can identify promising translational opportunities.
Targeted assays of ALG1 enzymatic function and unbiased screens to reverse phenotypes caused by loss of ALG1 in all disease models can be accomplished by a collaborative and distributed team of researchers who are project-managed well. Balance is advised between approaches to identify pharmacological chaperones that rescue unstable mutant ALG1 protein and approaches to identify disease modifiers upstream and downstream of ALG1.
ALG1-CDG patient fibroblasts have already been validated. From there drug repurposing screens offer the fastest and cheapest path to the first approved treatment for ALG1-CDG. Identify a bridge therapy that buys time for gene therapy and gene editing technologies to mature. In the case of ALG1 splice mutations, personalized antisense oligonucleotides (ASOs) offer hope. In parallel, replication of the proposed ALG1-CDG biomarker in Bengtson et al., 2016 is warranted.
Albright CF, Robbins RW. (1990). The sequence and transcript heterogeneity of the yeast gene ALG1, an essential mannosyltransferase involved in N-glycosylation. Journal of Biological Chemistry. 265: 7042-9.
Bengtson P, Ng BG, Jaeken J, Matthijs G, Freeze HH, Eklund EA. Serum transferrin carrying the xeno-tetrasaccharide NeuAc-Gal-GlcNAc2 is a biomarker of ALG1-CDG. (2016). Journal of Inherited Metabolic Disorders. 39: 107-114.
Grubenmann CE, Frank CG, Hülsmeier AJ, Schollen E, Matthijs G, Mayatepek E, Berger EG, Aebi M, Hennet T. (2004). Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik. Human Molecular Genetics. 13: 535-542.
Huffaker TC, Robbins PW. (1982). Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. Journal of Biological Chemistry. 257: 3203-3210.
Kranz C, Denecke J, Lehle L, Sohlbach K, Jeske S, Meinhardt F, Rossi R, Gudowius S, Marquardt T. (2004). Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I. American Journal of Human Genetics. 74: 545-51.
Ng BG, Shiryaev SA, Rymen D, Eklund EA, Raymond K, Kircher M, Abdenur JE, Alehan F, Midro AT, Bamshad MJ, Barone R, Berry GT, Brumbaugh JE, Buckingham KJ, Clarkson K, Cole FS, O’Connor S, Cooper GM, Van Coster R, Demmer LA, Diogo L, Fay AJ, Ficicioglu C, Fiumara A, Gahl WA, Ganetzky R, Goel H, Harshman LA, He M, Jaeken J, James PM, Katz D, Keldermans L, Kibaek M, Kornberg AJ, Lachlan K, Lam C, Yaplito-Lee J, Nickerson DA, Peters HL, Race V, Régal L, Rush JS, Rutledge SL, Shendure J, Souche E, Sparks SE, Trapane P, Sanchez-Valle A, Vilain E, Vøllo A, Waechter CJ, Wang RY, Wolfe LA, Wong DA, Wood T, Yang AC; University of Washington Center for Mendelian Genomics, Matthijs G, Freeze HH. (2016). ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients. Human Mutations. 37: 653-660.
Rexer TFT, Schildbach A, Klapproth J, Schierhorn A, Mahour R, Pietzsch M, Rapp E, Reichl U. (2018). One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides. Biotechnology and Bioengineering. 115: 192-205.
Schwarz M, Thiel C, Lübbehusen J, Dorland B, de Koning T, von Figura K, Lehle L, Körner C. (2004). Deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase causes congenital disorder of glycosylation type Ik. American Journal of Human Genetics. 74: 472-481.
Takahashi T, Honda R, Nishikawa Y. (2000). Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1. Glycobiology. 10: 321-327.
Valderrama-Rincon JD, Fisher AC, Merritt JH, Fan YY, Reading CA, Chhiba K, Heiss C, Azadi P, Aebi M, DeLisa MP. (2012). An engineered eukaryotic protein glycosylation pathway in Escherichia coli.Nature Chemical Biology. 8: 434-436.