The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in α-glucosidase I that give rise to congenital disorder of glycosylation IIb (CDG IIb)

Lec23 Chinese hamster ovary cells are defective in α-glucosidase I activity, which removes the distal α(1,2)-linked glucose residue from Glc₃Man₉GlcNAc₂ moieties attached to glycoproteins in the endoplasmic reticulum. Mutations in the human GCS1 gene give rise to the congenital disorder of glycosylation termed CDG IIb. Lec23 mutant cells have been shown to alter lectin binding and to synthesize predominantly oligomannosyl N-glycans on endogenous glycoproteins. A single point mutation (TCC to TTC; Ser to Phe) was identified in Lec23 Gcs1 cDNA and genomic DNA. Serine at the analogous position is highly conserved in all GCS1 gene homologues. A human GCS1 cDNA reverted the Lec23 phenotype, whereas GCS1 cDNA carrying the lec23 mutation (S440F in human) did not. By contrast, GCS1 cDNA with an R486T or F652L CDG IIb mutation gave substantial rescue of the Lec23 phenotype. Nevertheless, in vitro assays of each enzyme gave no detectable α-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates α-glucosidase I both in vitro and in vivo. However, the S440F α-glucosidase I may have a small amount of α-glucosidase I activity in vivo based on the low levels of complex N-glycans in Lec23. A sensitive test for complex N-glycans showed the presence of polysialic acid on the neural cell adhesion molecule. The Lec23 Chinese hamster ovary mutant represents a sensitive host for detecting a wide range of mutations in human GCS1 that give rise to CDG IIb.