CLONING AND EXPRESSION OF CDNA FOR A HUMAN GAL(BETA-1-3)GALNAC ALPHA-2,3-SIALYLTRANSFERASE FROM THE CEM T-CELL LINE

Complementary DNA encoding a human Gal(beta 1-3)GalNAc alpha 2,3-sialy ltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA li brary using a 23-base oligonucleotide probe. The sequence of this prob e was established on the basis of a slightly divergent sialylmotif L t hat was obtained by polymerase chain reaction with degenerate oligonuc leotide primers based on the conserved sialylmotif L of mammalian Gal( beta 1-3)GalNAc alpha 2,3-sialyltransferases. It was thus confirmed th at a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectiv ely 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J . C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% simil arity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, sk eletal muscle and various lymphoid tissues but not in brain and kidney . A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin a nd asialo-bovine submaxillary mucin appeared better substrates for hST 3Gal II than for its murine counterpart as previously reported [Kojima , N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Bioche mistry 33, 5772-5776]. In previous studies, we have shown hyposialylat ion of O-glycans attached to two major lymphocyte CD43 and CD45 cell s urface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-c ell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA an d enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection o f this cell line is probably due to a mechanism different from a simpl e altered catalytic activity of these sialyltransferases.