Search

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

Earlier studies reached conflicting conclusions as to the ability of the beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesizes gangliosides GM2 and GD2 to also produce gangliotriosylceramide (GA2). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain ganglioside GM3 as the most complex glycosphingolipid (GSL), whereas the CHO glycosylation mutant Lec2, which is deficient in sialylation, accumulates lactosylceramide with little GM3 being produced. We transfected both cell types with a plasmid containing a cloned GalNAc-T. Whereas transfected CHO cells produced GM2 as the major complex GSL, the major product in transfected Lec2 cells was GA2. Both types of transfected cells but not the untransfected cells expressed the transfected gene and contained high levels of enzyme activity for synthesizing both GM2 and GA2 in vitro. In summary, these results indicate that this enzyme can in fact synthesize GA2 as well as GM2 and GD2. In addition, these findings suggest that in CHO cells the synthesis of GM3 in vivo has priority over GA2 synthesis for utilization of the substrate lactosylceramide, resulting in little GA2 being produced even though GalNAc-T is present and active. Thus, competition for substrate between glycosylation pathways may have profound effects on the GSL pattern of cells.

We have utilized wheat germ agglutinin conjugated to iron/dextran particles in conjunction with high gradient magnetic affinity chromatography (HIMAC) to prepare plasma membranes from cultured cells. Membrane-impermeable succinimidyl esters inactivate alkaline phosphodiesterase 1 (APDE-1) and were used to establish the proportion of APDE-1 expressed at the cell surface. The yield of inhibitable APDE-1 provides an accurate indication of plasma membrane yield, which was > 90% for Chinese hamster ovary (CHO) cells. Plasma membranes prepared by HIMAC contained < 5-13% of endoplasmic reticulum, Golgi, mitochondria, lysosomes, or endosomes. Pulse-chase experiments performed with the alpha 5 beta 1 integrin receptor confirmed the high yield of plasma membrane and demonstrated the utility of this procedure for examining trafficking of proteins to and from the plasma membrane. We determined the lipid content of plasma membranes prepared by HIMAC. CHO plasma membranes contain 49% of total cellular phospholipid, 69% of sphingomyelin, and 64% of cholesterol. Phosphatidylserine was the only glycerophospholipid highly enriched (71%) in the retained fraction. The glycosphingolipids lactosylceramide and ganglioside GM3 were enriched in the plasma membrane fraction to the same extent as sphingomyelin. The major fraction of the glycosphingolipid precursors glucosylceramide and ceramide was localized to intracellular membranes. These findings indicate that the plasma membrane of CHO cells contains approximately half of the total cellular phospholipids and an even higher percentage of sphingomyelin and cholesterol. The high efficiency and rapidity of this isolation procedure should aid the analysis of plasma membrane components significantly.

Two monoclonal antibodies directed to the Type II carbohydrate chain of glycosphingolipids have been prepared by the murine hybridoma technique. Their reactivity was determined by liposome lysis, plate-binding assay, complement fixation, and hemagglutination inhibition. One antibody was specific for glycolipids having the nonreducing terminal N-acetyllactosamine (Gal beta 1 leads to 4GlcNAc beta 1 leads to R) structure and did not react with glycolipids having either a Type I chain (Gal beta 1 leads to 3GlcNAc beta 1 leads to R), a ganglio-series structure (Gal beta 1 leads to 3GalNAc beta 1 leads to R), or a sialosyl or fucosyl substitution at the N-acetyllactosamine residue. This antibody readily agglutinated adult human erythrocytes of all ABO types but did not agglutinate umbilical cord cells. The other antibody reacted with the Type II chain H structure (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to R) but reacted only weakly with the Type I chain H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to R) and ganglio-series H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GalNAc beta 1 leads to R) structures. At 37 degrees C this antibody agglutinated human erythrocytes of types O and A2 but was unable to cause detectable agglutination of types B and A1. Because of their narrow, well defined specificity, these monoclonal anticarbohydrate reagents will be useful in the study of the distribution, quantity, and function of specific carbohydrates on the cell surface.