Lec3 Chinese Hamster Ovary Mutants Lack UDP-N-acetylglucosamine 2-Epimerase Activity Because of Mutations in the Epimerase Domain of the Gne Gene

Abstract

Lec3Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced polysialic acid (PSA) on neural cell adhesion molecules. To identify the genetic origin of the phenotype, genes encoding different factors required for sialic acid biosynthesis were transfected into Lec3 cells. Only a Gne cDNA encoding UDP-GlcNAc 2-epimerase:ManNAc kinase rescued PSA synthesis. In an in vitro UDP-GlcNAc 2-epimerase assay, Lec3 cells had no detectable UDP-GlcNAc 2-epimerase activity, and Lec3 cells grown in serum-free medium were essentially devoid of sialic acid on glycoproteins. The Lec3 phenotype was rescued by exogenously added N-acetylmannosamine or mannosamine but not by the same concentrations of N-acetylglucosamine, glucosamine, glucose, or mannose. Sequencing of CHO Gne cDNAs identified a nonsense (E35stop) and a missense (G135E) mutation, respectively, in two independent Lec3 mutants. The G135E Lec3 mutant transfected with a rat Gne cDNA had restored in vitro UDP-GlcNAc 2-epimerase activity and cell surface PSA expression. Both Lec3 mutants were similarly rescued with a CHO Gne cDNA and with CHO Gne encoding the known kinase-deficient D413K mutation. However, cDNAs encoding the known epimerase-deficient mutation H132A or the new Lec3 G135E Gne mutation did not rescue the Lec3 phenotype. The G135E Gne missense mutation is a novel mechanism for inactivating UDP-GlcNAc 2-epimerase activity. Lec3 mutants with no UDP-GlcNAc 2-epimerase activity represent sensitive hosts for characterizing disease-causing mutations in the human GNE gene that give rise to sialuria, hereditary inclusion body myopathy, and Nonaka myopathy.

Mouse Large Can Modify Complex N- and Mucin O-Glycans on α-Dystroglycan to Induce Laminin Binding

“…This suggests that Large modifies N-glycans in Lec2 and Lec8 cells. Immunoblotting using a mAb against NCAM that is known to be N-glycosylated in CHO cells (64,65) showed that removal of N-glycans was efficiently achieved by the PNGase F treatment conditions used in this experiment. There was no effect of PNGase F on the ϳ38-kDa nonspecific band detected by IIH6.…”

A Mechanism of Sialylation Functionally Silences the Hyaluronan Receptor LYVE-1 in Lymphatic Endothelium

“…These included Lec1, which lacks N-acetylglucosaminyltransferase 1, required for the synthesis of complex, and hybrid N-glycans Lec2, which lacks CMP sialyltransferase required for terminal glycan sialylation (21), and Lec 3.2.8.1 (LecR) a compound mutant that lacks both these enzymes in addition to the UDP-N-acetylglucosamine-2-epimerase required for sialic acid biosynthesis and the UDP-galactose transporter (33,34). Importantly, each of the mutant CHO transfectants expressed comparable levels of LYVE-1 at the surface as assessed by LYVE-1 immunostaining and flow cytometry, and each showed a significant change in mobility relative to wild type during SDS-PAGE (Fig.…”

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)

“…5), although in this case, CHO cells clearly bound more anti-PSA mAb than Lec23 cells. Lec2 CHO cells that are deficient in CMP-sialic acid transport expressed almost no PSA as shown previously (27,45,46). S440F was engineered into a full-length human GCS1 cDNA, the mutant cDNA was unable to rescue the Lec23 phenotype, and transfectants had almost no detectable ␣-glucosidase I activity in vitro despite overexpression of the cDNA.…”