Lec23 Chinese hamster ovary cells are defective in
alpha-glucosidase I activity, which removes the distal alpha(1,2)-linked
glucose residue from Glc(3)
Man(9)GlcNAc(2) 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
alpha-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates
alpha-glucosidase I both in vitro and in vivo. However, the S440F
alpha-glucosidase I may have a small amount of
alpha-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.