The acceptor substrate specificity of human β4-galactosyltransferase V indicates its potential function in O-glycosylation

AbstractIn order to assess the function of the different human UDP-Gal:GlcNAc β4-galactosyltransferases, the cDNAs of two of them, β4-GalT I and β4-GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant β4-GalT V was more than 15 times lower than that of β4-GalT I, using GlcNAcβ-S-pNP as an acceptor. Whereas β4-GalT I efficiently acts on all substrates having a terminal β-linked GlcNAc, β4-GalT V appeared to be far more restricted in acceptor usage. β4-GalT V acts with high preference on acceptors that contain the GlcNAcβ1→6GalNAc structural element, as found in O-linked core 2-, 4- and 6-based glycans, but not on substrates related to N-linked or blood group I-active oligosaccharides. These results suggest that β4-GalT V may function in the synthesis of lacNAc units on O-linked chains, particularly in tissues which do not express β4-GalT I, such as brain

Regulation of fucosyltransferase-VII expression in peripheral lymph node high endothelial venules

Binding of L-selectin to the highly glycosylated peripheral lymph node addressins (PNAd) plays a central role in the normal recirculation of lymphocytes between the bloodstream and the lymph node. This interaction requires correct fucosylation of the PNAd, mediated by the recently identified fucosyltransferase-VII (Fuc-TVII). Here we show that during ontogeny Fuc-TVII is absent at the day of birth, barely detectable on day 1, and clearly present from day 2 onwards. PNAd expression as detected by the MECA-79 antibody precedes the expression of Fuc-TVII. Furthermore, we demonstrate that in adult mice antigenic stimulation of peripheral lymph nodes leads to a temporary disappearance of Fuc-TVII at days 2 and 3 after stimulation, followed by a complete reappearance by day 4, while expression of MECA-79 is never completely absent during this period. Finally, occlusion of afferent lymphatics to peripheral lymph nodes resulted in a decreased expression of Fuc-TVII in the high endothelial venules by day 5, and complete disappearance within 8 days. We conclude that the activity of Fuc-TVII in cells of high endothelial venules is directly affected by afferent lymph and activation processes that occur in the lymph node after antigenic stimulation. The expression of Fuc-TVII is therefore yet another level at which the function of high endothelial venules, and thus lymphocyte trafficking, can be regulated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48706/1/3040_ftp.pd

Remodeling of the Carbohydrate Chains of hCG by Use of Sialyltransferases: Effects on the Biological Activity

Human chorionic gonadotropin (hCG) is a glycoprotein hormone which contains both N- and O-linked carbohydrate chains. It consists of two subunits, a and 8. The a-subunit contains two N-linked carbohydrate chains: a mono-antenna and a non-fucosylated bi-antenna. The $-subunit contains both (two) N and (four) O-linked carbohydrate structures. Both of the N-linked carbohydrate chainsare biantennary, one of which is fucosylated

a1>3-GALACTOSYLTRANSFERASE : THE USE OF RECOMBINANT ENZYME FOR THE SYNTHESIS OF a-GALACTOSYLATED GLYCOCONJUGATES

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDPGal: Galß1>4G1cNAc «1>3-galactosyltransferase (Joziasse, D.H. et al., (1989) J. Biol. Chem. 264, 14290-14297). Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Sf9 insect cells with recombinant virus, resulted in high-level expression of enzymatically active al>3-galactosyltransferase. The recombinant &1>3-galactosyltransferase could be readily detergent solubilized and subsequently purified by affinity-chromatography on UDP-hexanolamine-Sepharose. The recombinant «1>3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (GalB1>4G1cNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus a1 >3- galactosyltransferase

SIALYLTRANSFERASES; THEIR SPECIFICITY AND THEIR USE IN CARBOHYDRATE REMODELLING.

In order to apply sialyltransferases in the remodelling of the carbohydrate chains on biologically active glycoproteins, it is a prerequisite to know the fine specificity of these enzymes. In this report the specificity of several sialyltransferses involved in the sialylation of O- and N-linked oligosaccharide chains is reviewed. Also novel results on the branch specificity of a3- and a6-sialyltransferase are reported. The potential application of these enzymes in carbohydrate remodelling was studied using human chorionic gonadotropin (hCG) as a model glycoprotein. Differently sialylated preparations of this hormone were obtained and tested for their stimulatory effect on steroidogenesis in Leydig cells in vitro. Asialo-hCcG appeared to be only 45% as effective as native hCG. a3-Resialylation of the O-linked chains on the ß-subunit of this hormone did not restore the biological activity to a higher level. By contrast, 55% a6- resialylation of the N-linked chains yielded a preparation which was almost as active as native hCG. Interestingly, further sialylation by the a6-sialyltransferase resulted in a decrease of the bio-activity to levels lower than obtained with asialo-hcG. It is concluded that the lectin-carbohydrate binding, which is part of the process that triggers the biological respons of the target cell can be mimicked by N-linked chains carrying a6-linked sialic acid. However, too high a density of such residues interferes with this interaction