Abstract

Endo-beta-galactosidase from Escherichia freundii has been useful for structural and immunochemical analysis of cell surface antigens such as Ii, ABH, F9, and SSEA-1. Further development of endo-beta-galactosidase will provide an indispensable tool in the analysis of the role of cell-surface carbohydrate chains in differentiation and tumorigenicity. Expecting that endo-beta-galactosidase from different origins has substrate specificities different from each other, endo-beta-galactosidases found in Diplococcus pneumoniae and Bacterioides fragilis have been purified and characterized. The former enzyme is found to be unique in that the enzyme hydrolyzes the lactosaminoglycan (LAG) by stepwise endoglycosidic action. The latter was found to be similar to E. freundii enzyme. Since antigens Ii, F9, and SSEA-1 are specifically expressed at different stages of mouse embryonal differentiation, glycoconjugate profiles of cell surfaces were analyzed by using mouse terato-carcinoma cell lines as a model system. """"""""Nullipotent"""""""" embryonal carcinoma cell line (F9) was stimulated to differentiate into parietal endoderm in the presence of retinoic acid and dibutyryl cyclic AMP or into visceral endoderm in the presence of retinoic acid. Surface glycoconjugates of each stage of cells were analyzed as to whether each component has polylactosamino structure characterized by susceptibility to endo-beta-galactosidase. In addition, LAG was isolated from human embryonal carcinoma cell line PAl. The structure of embryonal LAG was analyzed by endo-beta-galactosidase treatment, methylation analysis, and fast atom bombardment mass spectrometry. PAl lactosaminoglycans were found to have new disialosylterminal NeuAcd2 9NeuAcd2 3/6Gal, which is expected to be specific to early embryo. (A)

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034014-03
Application #
3171744
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1983-02-01
Project End
1986-06-30
Budget Start
1985-02-01
Budget End
1986-06-30
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
009214214
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

Aoki, D; Appert, H E; Johnson, D et al. (1990) Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. EMBO J 9:3171-8
Fukuda, M N; Masri, K A; Dell, A et al. (1990) Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding alpha-mannosidase II. Proc Natl Acad Sci U S A 87:7443-7
Fukuda, M N; Masri, K A; Dell, A et al. (1989) Defective glycosylation of erythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of low level of membrane-bound form of galactosyltransferase. Blood 73:1331-9
Masri, K A; Appert, H E; Fukuda, M N (1988) Identification of the full-length coding sequence for human galactosyltransferase (beta-N-acetylglucosaminide: beta 1,4-galactosyltransferase). Biochem Biophys Res Commun 157:657-63
Fukuda, M N; Dell, A; Scartezzini, P (1987) Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II. J Biol Chem 262:7195-206
Howard, D R; Fukuda, M; Fukuda, M N et al. (1987) The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates. J Biol Chem 262:16830-7
Izhar, M; Siebert, P D; Oshima, R G et al. (1986) Trophoblastic differentiation of human teratocarcinoma cell line HT-H1. Dev Biol 116:510-8
Fukuda, M N; Bothner, B; Lloyd, K O et al. (1986) Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence. J Biol Chem 261:5145-53
Fukuda, M N; Klier, G; Yu, J et al. (1986) Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II. Blood 68:521-9
Fukuda, M N; Bothner, B; Scartezzini, P et al. (1986) Isolation and characterization of poly-N-acetyllactosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients. Chem Phys Lipids 42:185-97

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