Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.     

The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-O-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galbeta1,3GalNAc alpha-O-Al to act as an acceptor for Group A. (2) An alpha1,3-fucosyl residue on the beta1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the beta1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta1,3 to GalNAc alpha. (5) Group A activity on Gal linked beta1,3 to GalNAc remained unaffected by 3'-sulfation of the beta1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galbeta1,4GlcNAcbeta-O-Al and Galbeta1,3GlcNAcbeta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as acceptors for Group B.     

Administration of a crystalloid fluid preload does not prevent the decrease in arterial blood pressure after induction of anaesthesia with propofol and fentanyl

Five oligosaccharide alpha1-phosphates and one sulfated glycopeptide have been isolated from the hemofiltrate of one patient with end-stage renal disease. Isolation of these compounds has been achieved using reverse osmosis, ion-exchange and size-exclusion chromatography and high performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H and 31P NMR spectroscopy. The chemical structures were determined to be: 1 NeuAc alpha2-3Gal alpha1-OPO3H2; 2 NeuAc alpha2-6Galbeta1-4GlcNAc alpha1-OPO3H2; 3 NeuAc alpha2-3Galbeta1-3GalNAc alpha1-OPO3H2; 4 NeuAc alpha2-3Galbeta1-3[NeuAc alpha2-6]GalNAc alpha1-OPO3H2 (proposed structure); 5 Fuc alpha1-2Galbeta1-4[Fuc alpha1-3]GlcNAc alpha1-OPO3H2; 6 HOSO3-4Fuc alpha1-6GlcNAcbeta1-NAsn. While 2 and 3 have been previously characterized as compounds of urine and hemofiltrate, the oligosaccharide alpha1-phosphates 1, 4, and 5 could be isolated--to our knowledge--for the first time from biological material. Compound 6 is the first glycopeptide reported to contain a 4-sulfated fucose residue.     

Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-->R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.     

Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1-->6GalNAcalpha core structure as a tumor-associated antigen

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.     

Expression of human H-type alpha1,2-fucosyltransferase encoding for blood group H(O) antigen in Chinese hamster ovary cells. Evidence for preferential fucosylation and truncation of polylactosamine sequences

The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.     

Selective repopulation of normal mouse liver by Fas/CD95-resistant hepatocytes

The oligosaccharide sequences of glycoconjugates in the human normal epididymis and the nature of linkages were studied with lectin histochemistry. The usual terminal sequences of oligosaccharide side chains in epithelial cell secretions were Neu5Ac2,3Galbeta1,3GalNAc; SO4Galbeta1,3GalNAc; and Galbeta1,4GlcNAc, and they were mainly found in O-linked glycoproteins. The lectin pattern of mitochondria-rich cells differed from that of principal cells.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)-GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.     

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells

Beta1,4-galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of beta1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 3) Manbeta1-4GlcNAcbeta1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1)beta1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity.     

Stable expression of the Golgi form and secretory variants of human fucosyltransferase III from BHK-21 cells. Purification and characterization of an engineered truncated form from the culture medium

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha1, 3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galbeta1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta1-3GlcNAc. The type II motif Galbeta1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha2-3/alpha2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.     

Structural changes in the oligosaccharide moiety of human IgG with aging

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of +/-Galbeta1-4GlcNAcbeta1-2Manalpha1-6(+/-GlcNAcbeta 1-4)(+/-Galbeta1-4GlcNAcbeta1-2Man(alpha)1-3)Man(beta)1-+ ++4GlcNAcbeta1-4(+/- Fucalpha1-6)GlcNAc and their mono- and disialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<-0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r=0.461 and r= -0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.     

Molecular cloning of three sulfotransferase cDNAs from mouse liver

Sulfate conjugation plays an important role in the biotransformation of not only xenobiotics but also many endogenous substances. Sulfotransferases, the enzymes that are responsible for this process, exist as a superfamily of genes. It has long been recognized that significant species differences exist among drug and carcinogen metabolizing enzymes such as cytochrome P450. Species differences in both regulation and catalytic activities of sulfotransferases may also exist. To investigate this, we conducted cDNA cloning and cDNA expression studies of sulfotransferase in the mouse. Three sulfotransferase cDNA clones were isolated from a female B6CBA mouse liver. Two of the clones, mSTa1 and mSTa2, were highly homologous to each other. Alignment of mSTa1 and mSTa2 cDNAs' nucleotide sequences with those of other sulfotransferase cDNAs revealed the greatest sequence identity with the rat STsmp cDNA. This analysis suggests that mSTa1, mSTa2 and rSTsmp cDNAs are derived from orthologous genes belonging to the alcohol/hydroxysteroid sulfotransferase gene family. The third clone, mSTp1 showed high identity to rSTp, hSTp1, hSTp3, and rSTp1C1, suggesting that mSTp1 belongs to the phenol family.     

Neisseria gonorrhoeae that infect men have lipooligosaccharides with terminal N-acetyllactosamine repeats

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2(+) and 06B4(+)) and GalNAc-->lacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta-N-acetylhexosaminidase and beta-galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta1-->4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.     

Galalpha1-->4Gal-glycans are expressed on myofibrillar associated proteins

There is evidence that glycans carrying terminal galactose residues are differently expressed in the sarcoplasm of different muscle fiber types. In this study monoclonal antibodies directed against P blood group antigens Pk: Galalpha1-4Galbeta1-4Glcbeta- and P1: Galalpha1-4Galbeta1-4GlcNAcbeta- were used to detect terminal alpha-galactosylated glycoconjugates on muscle proteins. Electrotransfer of proteins, extracted from human masseter and biceps muscles, to nitrocellulose after polyacrylamide gel electrophoresis (PAGE) and incubation with anti-Pk (CD77) consistently showed two bands with apparent molecular weights of 66 kDa and 64 kDa. In fresh frozen muscle sections from some humans there was endothelial reaction with anti-CD77 in capillaries, venules and veins but not in arterioles and arteries. In muscle samples from other humans there was no staining of endothelial cells. Formalin-fixed human muscle displayed a CD77 reaction with highest accumulation of reaction product at the periphery of the fibers. This may be explained by the presence of Pk glycoconjugates on intermediate filaments in muscle fibers. In preparations of cat masseter muscle proteins the antibodies against P1Pk antigens reacted with a 170 kDa and a 55 kDa band while in preparations of cat biceps brachii only a 55 kDa band was reactive. The specificities of the antibodies were investigated by fluorescence-activated cell sorter (FACS), alpha- and beta-galactosidase digestion and inhibitory sugars. This study indicates that glycans carrying Galalpha1-4Galbeta1- epitopes are expressed on myofibrillar associated proteins.     

Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains

A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.