Analysis of the specificity of five murine anti-blood group A monoclonal antibodies, including one that identifies type 3 and type 4 A determinants

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174

161

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 g of VT-2 or 1.0 g of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

Section: Methodsmentioning

confidence: 99%

Targeted Disruption of Gb3/CD77 Synthase Gene Resulted in the Complete Deletion of Globo-series Glycosphingolipids and Loss of Sensitivity to Verotoxins

Okuda

Tokuda

Numata

et al. 2006

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161

“…Glycolipid Extraction-Glycolipids were extracted as described previously (23). For TLC immunostaining, a neutral glycolipid fraction derived from 0.25 ml of each blood sample was spotted on each lane.…”

Section: Methodsmentioning

confidence: 99%

The Blood Group P1 Synthase Gene Is Identical to the Gb3/CD77 Synthase Gene

Iwamura

Furukawa

Uchikawa

et al. 2003

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Blood group P1/P2 is a glycolipid antigen system for which the genetic mechanism has not yet been clarified. We analyzed the potential of the cloned Gb3/CD77 synthase to synthesize P1 antigen, because Gb3/CD77 and P1 share a common structure, Gal␣1,4Gal␤1,4Glc (NAc)؊. L cell transfectants with Gb3/CD77 synthase cDNA expressed marginal levels of P1 on the cell surface but contained high levels of P1 in the cytoplasm. P2-type erythrocytes, which were serotyped as P2, also contained definite P1 antigen inside cells, although the amounts were lower than those of P1 cells. Only p erythrocytes lacked P1 antigen corresponding with functionlosing mutations in the Gb3/CD77 synthase gene. Synthesis of P1 antigen from paragloboside in vitro was demonstrated using membrane fraction of the transfectants and a fusion enzyme with protein A. These results strongly suggested that P1 synthase is identical to Gb3/ CD77 synthase and appear to propose a clue for the solution of the long-pending P1/P2/p puzzle. The P1/P2 difference might result from the difference in P1 quantity based on either different enzyme activity or the presence/absence of other enzyme modulators. Because P2 erythrocytes showed lower levels of Gb3/CD77 synthase mRNA than P1, 5-upstream promoter regions were analyzed, resulting in the identification of two P2-specific homozygous mutations. Differences in the transcriptional regulation in erythrocytes might be a major factor determining P1/P2.

“…Genetic typing of the GM2/GD2 synthase gene was performed as described previously (11). Extraction of Glycolipids-Glycolipids were extracted as described previously (18). Briefly, lipid fractions were extracted by chloroform/ methanol at ratios of 2:1, 1:1, and 1:2, sequentially.…”

Section: Methodsmentioning

confidence: 99%

Refractory Skin Injury in Complex Knock-out Mice Expressing Only the GM3 Ganglioside

Inoue

Fujii

Furukawa

et al. 2002

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108

We generated double knock-out mice lacking the GM2/ GD2 and the GD3 synthase gene by mating single gene mutants, and we analyzed the abnormal phenotypes of the mutant mice expressing only the GM3 ganglioside. We observed a refractory skin lesion that appeared primarily on the face of the mutant mice at 25 weeks after birth or later. Frequent scratching of the wound sites was observed in mutant mice with the skin injury, suggesting that it is a triggering factor that exacerbates the injury. This was confirmed by isolating mice in special cages for metabolic study in which the skin injury developed more rapidly. Characteristic proliferation of nerve fibers was found in the epidermis and subepidermis at the injured sites of the mutants, probably a result of continuous skin injury. Peripheral nerve degeneration was observed in young mutant mice, suggesting that reduced sensory function induced over-scratching and the resulting skin lesion. The fact that sensory response to mechanical stimuli decreased while that to hot stimuli increased in the mutant mice supports this interpretation. Thus, only GM3-expressing mice displayed the important role of gangliosides in maintaining skin integrity via regulation of the peripheral nerves.Acidic glycosphingolipids, gangliosides, are ubiquitously expressed in the various tissues of vertebrates and play important roles in the regulation of highly organized multicellular systems (1). In particular, they are very much enriched in the nervous system and have been considered to control development, proliferation, differentiation, and maintenance of the neural tissues and cells (2, 3). Expression patterns of various ganglioside species in the nervous system vary during development and are strictly regulated in a spatio-temporal manner (4), suggesting that individual structures of gangliosides contain significant implications for individual situations.A number of studies have been performed to demonstrate the biological function of gangliosides primarily by modifying their structures with enzymes or using inhibitors to block some steps of synthesis (5). Otherwise, the effects of addition of gangliosides to the culture medium or injection into the conditioned animals have been the primary approaches in addressing the significance of gangliosides. However, recent success in the molecular cloning of glycosyltransferase genes brought about an evolutional change in the experimental approaches for carbohydrate function analysis. Availability of glycosyltransferase genes responsible for the synthesis of gangliosides enabled us to remodel ganglioside compositions of cells and tissues in vivo (6, 7) and in vitro (8).Because we isolated GM2/GD2 synthase (EC 2.4.1.92) cDNA (9) and GD3 synthase (EC 2.4.99.8) cDNA (10), we established gene knock-out mice lines of the individual glycosyltransferases. GM2/GD2 synthase gene knock-out mice lacking all complex gangliosides showed almost normal morphogenesis of the nervous system and no definite abnormal behaviors (11), although they showed nerve degene...