During fertilization in mice, free-swimming sperm bind to mZP3, an 83-kDa glycoprotein present in the egg extracellular coat, the zona pellucida [Wassarman, P. M. (1990) Development 108, 1-17]. Mouse sperm recognize and bind to a specific class of serine/threonine-linked (O-linked) oligosaccharides present on mZP3. After binding to mZP3, sperm undergo a form of cellular exocytosis, the acrosome reaction, thereby enabling them to penetrate the zona pellucida and fertilize the egg. Thus, gamete interactions in mice are carbohydrate-mediated. In this context, we tested 15 O-linked-related oligosaccharide constructs with defined structures for their ability to inhibit binding of mouse sperm to ovulated eggs and to induce sperm to undergo the acrosome reaction in vitro. Thirteen of the oligosaccharides were constructed and characterized in our laboratory [Seppo, A., Pentillä, L., Niemelä, R., Maaheimo, H., Renkonen, O., & Keane, A. (1995) Biochemistry 34, 4655-4661]; two were obtained commercially. We found that, while none of the oligosaccharides induced sperm to undergo the acrosome reaction, a few of them inhibited binding of sperm to eggs at relatively low concentrations (ID50 < 5 microM). In certain cases, sperm formed head-to-head aggregates in the presence of the oligosaccharides. The results suggest that the ability of oligosaccharides to inhibit binding of sperm to eggs is dependent on several parameters, including the size and branching pattern of the oligosaccharide, as well as on the nature of the sugar residue at the nonreducing end of the oligosaccharide.

The P-selectin counterreceptor PSGL-1 is covalently modified by mono ␣2,3-sialylated, multiply ␣1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte ␣1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of ␣2,3-sialylated acceptors but is ineffective in transfer to the distal ␣2,3-sialylated LN unit in ␣2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal ␣2,3-sialylated LN unit in ␣2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SA␣2-3LN␤1-3LN␤1-3LN (where SA is sialic acid) into the trifucosylated molecule SA␣2-3Lex␤1-3Lex␤1-3Lex (where Lex is the trisaccharide Gal␤1-4(Fuc␣1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.

We show here that colon-carcinoma cell lines adhere to E-selectin via sialyl Lewis x and sialyl Lewis a (s(Lex) and s(Lea)) oligosaccharides and that this adhesion can be enhanced by TNF stimulation. To study in greater detail this endothelial binding, we analysed the mRNA expression and function of the enzymes participating in the generation of s(Lex) and s(Lea on cancer cells. These oligosaccharides are synthesized by sequential action of alpha 2,3 sialyl (alpha 2,3-ST) and alpha 1,3/1/4 fucosyltransferases (alpha 1,3/1,4-FT) on existing (poly)N-acetyllactosamine chains. We report here that mRNAs of 2 recently cloned alpha 2,3-STs and 4 alpha 1,3/1,4-FTs are expressed in adenocarcinoma cells. In functional assays alpha 2,3-ST and alpha 1,3- or 1,4-FT activities were observed in adenocarcinoma cell lysates to exogenous N-acetyllactosamine and lacto-N-biose acceptors and to their sialylated derivatives, leading to the synthesis of the sialyl-N-acetyllactosamine and s(Lex) or the sialyllacto-N-biose and s(Lea), respectively. Furthermore, the inflammatory cytokine TNF could enhance some alpha 2,3-ST and alpha 1,3/1,4-FT activities capable of generating E-selectin counter-receptors. Taken together, these data show that COLO 205 and HT-29 adenocarcinoma cell lines adhere to E-selectin in a TNF-inducible manner via their cell-surface s(Lex) and s(Lea). These cells also express mRNA as well as inducible enzyme activities of several alpha 2,3-STs and alpha 1,3/1,4-FTs responsible for the final steps in the synthesis of s(Lex) and s(Lea).(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel linear tetrasaccharide, Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc beta 1-6Gal beta 1-4GlcNAc, resisted the action of endo-beta-galactosidase (EC 3.2.1.103) from E. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc were cleaved in the expected manner.