Search

Complex and hybrid N-glycans contain sugar residues that have been implicated in fertilization, compaction of the embryo, and implantation. Inactivation of the Mgat1 gene responsible for their synthesis is embryonic lethal, but homozygous mutant blastocysts are phenotypically normal due to the presence of maternal Mgat1 gene transcripts. To identify roles for complex and hybrid N-glycans in oogenesis and preimplantation development, the Mgat1 gene in oocytes was deleted by using a ZP3Cre recombinase transgene. All mutant oocytes had an altered zona pellucida (ZP) that was thinner than the control ZP, and they did not possess complex N-glycans but contained ZP1, ZP2, and ZP3 glycoproteins. Mutant eggs were fertilized, all embryos implanted, and heterozygotes developed to birth. However, mutant females had decreased fertility, yielded fewer eggs after stimulation with gonadotropins, and produced a reduced number of preimplantation embryos and less progeny than controls. About 25% of embryonic day 3.5 (E3.5) embryos derived from mutant eggs were severely retarded in development, even when they were heterozygous and expressed complex N-glycans. Thus, a proportion of Mgat1(-)(/)(-) oocytes were developmentally compromised. Surprisingly, mutant eggs also gave rise to Mgat1(-)(/)(-) embryos that developed normally, implanted, and progressed to E9.5. Therefore, complex or hybrid N-glycans are required at some stage of oogenesis for the generation of a developmentally competent oocyte, but fertilization, blastogenesis, and implantation may proceed in their absence.

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.

Three distinct Chinese hamster ovary mutants selected for resistance to wheat germ agglutinin were previously described by this laboratory. In this paper, evidence is provided that each phenotype occurs at a similar frequency in an unmutagenized population of Chinese hamster ovary cells. Two novel wheat germ agglutinin resistance phenotypes (WgaR), which also appear to occur at similar frequencies were uncovered in the course of these studies. One mutant type belongs to a new, recessive complementation group (VIII), and the second belongs to a previously defined complementation group (VI). Mutants from each of the four WgaR complementation groups (I, II, III, and VIII) exhibited characteristic and unique patterns of resistance to the toxicity of a variety of plant lectins. These properties were used in developing independent selection protocols which were highly specific for the isolation of each of the mutant types.

To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.