Asn-linked oligosaccharides in lectin-resistant tumor-cell mutants with varying metastatic potential.

Abstract

MDW4, a wheat germ agglutinin-resistant mutant of the metastatic murine tumor MDAY D2 has previously been shown to be poorly metastatic when injected intravenously and non-metastatic when injected subcutaneously into syngeneic mice. W4EB8, a Bandeiraea simplicifolia (BSII) lectin-selected subline of MDW4 has previously been shown to be intermediate between that of MDAY-D2 and MDW4 cell for sensitivity to lectin and metastatic phenotype when injected intravenously into mice. The Asn-linked oligosaccharides from MDAY-D2, MDW4 and W4EB8 cells were released enzymatically with peptide N-glycosidase, reduced with tritiated sodium borohydride and fractionated by Concanavalin-A--Sepharose affinity chromatography and high-performance liquid chromatography (HPLC). Structures of the major fractions were determined by a combination of glycosidase digestion and sizing, gas-liquid chromatography/mass spectrometry and by proton nuclear magnetic resonance. Wild-type and mutant cells processed high-mannose-type structures to biantennary (GlcNAc)2(Man)3(GlcNAc)2. In MDAY-D2 cells this structure was processed further to sialylated tetra-antennary complex with polylactosamine-containing antennae terminating in either sialic acid or alpha 1-3-linked galactose. MDW4 cells had four or five times more (GlcNAc)2(Man)3(GlcNAc)2 than MDAY-D2 cells and a major component of tri-antennary (GlcNAc)3(Man)3(GlcNAc)2 (i.e. 2,2,6-substituted tri-mannosyl core) that was not found in wild-type cells. The partial revertant, W4EB8 had intermediate levels of mutant (GlcNAc)3(Man)3(GlcNAc)2 and sialylated complex-type carbohydrates. The results indicate that a shift in expression from incomplete complex type to sialylated tri/tetra-antennary complex-type carbohydrates in tumor cell may enhance the metastatic potential of tumor cells in the experimental metastasis assay. In addition, somatic cell hybridization analysis indicated that the defect in MDW4 cells was identical to that of the Chinese hamster ovary mutant Lec8: a deficiency in UDP-galactose transport into the golgi.