Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Dec 19;92(26):12070–12074. doi: 10.1073/pnas.92.26.12070

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

T Hennet 1, F K Hagen 1, L A Tabak 1, J D Marth 1
PMCID: PMC40298  PMID: 8618846

Abstract

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

Full text

PDF
12070

Images in this article

12072Fig. 1
on p.12072
12073Fig. 2
on p.12073
12073Fig. 3
on p.12073

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baubonis W., Sauer B. Genomic targeting with purified Cre recombinase. Nucleic Acids Res. 1993 May 11;21(9):2025–2029. doi: 10.1093/nar/21.9.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chaffin K. E., Beals C. R., Wilkie T. M., Forbush K. A., Simon M. I., Perlmutter R. M. Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. EMBO J. 1990 Dec;9(12):3821–3829. doi: 10.1002/j.1460-2075.1990.tb07600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fenderson B. A., Eddy E. M., Hakomori S. Glycoconjugate expression during embryogenesis and its biological significance. Bioessays. 1990 Apr;12(4):173–179. doi: 10.1002/bies.950120406. [DOI] [PubMed] [Google Scholar]
  4. Fukushige S., Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7905–7909. doi: 10.1073/pnas.89.17.7905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Golic K. G., Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell. 1989 Nov 3;59(3):499–509. doi: 10.1016/0092-8674(89)90033-0. [DOI] [PubMed] [Google Scholar]
  6. Gu H., Marth J. D., Orban P. C., Mossmann H., Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science. 1994 Jul 1;265(5168):103–106. doi: 10.1126/science.8016642. [DOI] [PubMed] [Google Scholar]
  7. Gu H., Zou Y. R., Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993 Jun 18;73(6):1155–1164. doi: 10.1016/0092-8674(93)90644-6. [DOI] [PubMed] [Google Scholar]
  8. Hagen F. K., Van Wuyckhuyse B., Tabak L. A. Purification, cloning, and expression of a bovine UDP-GalNAc: polypeptide N-acetyl-galactosaminyltransferase. J Biol Chem. 1993 Sep 5;268(25):18960–18965. [PubMed] [Google Scholar]
  9. Homa F. L., Hollander T., Lehman D. J., Thomsen D. R., Elhammer A. P. Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem. 1993 Jun 15;268(17):12609–12616. [PubMed] [Google Scholar]
  10. Kilby N. J., Snaith M. R., Murray J. A. Site-specific recombinases: tools for genome engineering. Trends Genet. 1993 Dec;9(12):413–421. doi: 10.1016/0168-9525(93)90104-p. [DOI] [PubMed] [Google Scholar]
  11. Kishihara K., Penninger J., Wallace V. A., Kündig T. M., Kawai K., Wakeham A., Timms E., Pfeffer K., Ohashi P. S., Thomas M. L. Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell. 1993 Jul 16;74(1):143–156. doi: 10.1016/0092-8674(93)90302-7. [DOI] [PubMed] [Google Scholar]
  12. Kleene R., Berger E. G. The molecular and cell biology of glycosyltransferases. Biochim Biophys Acta. 1993 Dec 21;1154(3-4):283–325. doi: 10.1016/0304-4157(93)90003-7. [DOI] [PubMed] [Google Scholar]
  13. Lakso M., Sauer B., Mosinger B., Jr, Lee E. J., Manning R. W., Yu S. H., Mulder K. L., Westphal H. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232–6236. doi: 10.1073/pnas.89.14.6232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lowe J. B., Stoolman L. M., Nair R. P., Larsen R. D., Berhend T. L., Marks R. M. ELAM-1--dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell. 1990 Nov 2;63(3):475–484. doi: 10.1016/0092-8674(90)90444-j. [DOI] [PubMed] [Google Scholar]
  15. McEver R. P., Moore K. L., Cummings R. D. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem. 1995 May 12;270(19):11025–11028. doi: 10.1074/jbc.270.19.11025. [DOI] [PubMed] [Google Scholar]
  16. Nagy A., Rossant J., Nagy R., Abramow-Newerly W., Roder J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8424–8428. doi: 10.1073/pnas.90.18.8424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ong C. J., Chui D., Teh H. S., Marth J. D. Thymic CD45 tyrosine phosphatase regulates apoptosis and MHC-restricted negative selection. J Immunol. 1994 Apr 15;152(8):3793–3805. [PubMed] [Google Scholar]
  18. Orban P. C., Chui D., Marth J. D. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6861–6865. doi: 10.1073/pnas.89.15.6861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Reisner Y., Linker-Israeli M., Sharon N. Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. Cell Immunol. 1976 Jul;25(1):129–134. doi: 10.1016/0008-8749(76)90103-9. [DOI] [PubMed] [Google Scholar]
  20. Roth J., Wang Y., Eckhardt A. E., Hill R. L. Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8935–8939. doi: 10.1073/pnas.91.19.8935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sastry M. V., Banarjee P., Patanjali S. R., Swamy M. J., Swarnalatha G. V., Surolia A. Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc). J Biol Chem. 1986 Sep 5;261(25):11726–11733. [PubMed] [Google Scholar]
  22. Sutherland D. R., Abdullah K. M., Cyopick P., Mellors A. Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica. J Immunol. 1992 Mar 1;148(5):1458–1464. [PubMed] [Google Scholar]
  23. True D. D., Singer M. S., Lasky L. A., Rosen S. D. Requirement for sialic acid on the endothelial ligand of a lymphocyte homing receptor. J Cell Biol. 1990 Dec;111(6 Pt 1):2757–2764. doi: 10.1083/jcb.111.6.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wang Y., Agrwal N., Eckhardt A. E., Stevens R. D., Hill R. L. The acceptor substrate specificity of porcine submaxillary UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is dependent on the amino acid sequences adjacent to serine and threonine residues. J Biol Chem. 1993 Nov 5;268(31):22979–22983. [PubMed] [Google Scholar]
  25. White T., Bennett E. P., Takio K., Sørensen T., Bonding N., Clausen H. Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem. 1995 Oct 13;270(41):24156–24165. doi: 10.1074/jbc.270.41.24156. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES