Mechanisms of Expression of a Burkitt Lymphoma-Associated Antigen (Globotriaosylceramide) in Burkitt Lymphoma and Lymphoblastoid Cell Lines

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.

Section: Discussionsupporting

confidence: 52%

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

Iwamura

Furukawa

Uchikawa

et al. 2003

“…Expression of Gb3/CD77 antigen is sometimes cryptic, depending on co-expressed sialosyl components on the cell surface (29). The SK-MEL-37 melanoma line, which was the source of the cDNA library used, was also negative for CD77 expression despite showing a definite ␣1,4Gal-T activity.…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning of Globotriaosylceramide/CD77 Synthase, a Glycosyltransferase That Initiates the Synthesis of Globo Series Glycosphingolipids

Kojima¹,

Fukumoto²,

Okajima³

et al. 2000

131

120

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (␣1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed ␣1,4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

“…It seemed possible that the reduction of Gb3/CD77 expression was caused by increased crypticity due to co-expressed sialylglycoconjugates as previously described (25). However, sialidase treatment of MEG-01 did not affect so much the expression levels of Gb3/CD77 as well as of GPIIb-IIIa at both TPA-treated and untreated conditions.…”

Section: Discussionmentioning

confidence: 60%

“…In particular, EB-B cells exhibited fairly high expression levels of Gb3/ CD77 synthase gene, while Gb3/CD77 expression is minimal or null. This may be due to the crypticity of the antigen (25) or the lack of lactosylceramide, and remains to be clarified. This is the first study to detect Gb3/CD77 expression on megakaryoblasts, although the finding that human platelets contain a high level of Gb3/CD77 as a major glycolipid is well known (26 -28).…”

Section: Discussionmentioning

confidence: 99%

Expression of the Gb3/CD77 Synthase Gene in Megakaryoblastic Leukemia Cells

Yokoyama

Sato

Wiels

et al. 2002

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the ␣1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.