Biomedical subjects

R D Cummings

Publications and source records attributed to R D Cummings.

At least 91 records · Page 5Linked to original sources

PubMed1992

The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid.

Previous studies have shown that the glycoprotein oligosaccharides synthesized by adult Schistosoma mansoni, the organism responsible for human schistosomiasis, are unusual in that they contain terminal beta-GalNAc residues and lack sialic acid. These observations and other studies indicating that schistosome glycoproteins and glycolipids are antigenic in infected animals led us to investigate the structures of the glycosphingolipids synthesized by these organisms and to determine whether they are structurally related to those synthesized by their vertebrate hosts. For our studies, adult schistosomes were metabolically radiolabeled with either [3H]galactose or [3H]glucosamine, and the newly synthesized glycosphingolipids were isolated and characterized. The major glycosphingolipids synthesized by adult schistosomes were found to be galactosylceramide and glucosylceramide. The adult worms synthesized no lactosylceramide (Gal beta 1-4Glc-ceramide), a common constituent of vertebrate cells; however, another disaccharide-containing glycosphingolipid cleavable by ceramide glycanase was found. The results of compositional and methylation analyses and exoglycosidase treatments demonstrated that this ceramide-disaccharide has the structure GalNAc beta 1-4Glc-ceramide. We also found that extracts of adult schistosomes are unable to transfer Gal from UDP-Gal to glucosylceramide, whereas extracts of Chinese hamster ovary cells, as a control, are able to do so, confirming that schistosomes are unable to synthesize lactosylceramide. Low levels of higher molecular weight glycosphingolipids were also found to be synthesized by adult schistosomes, and although their levels were too small to allow definitive characterization, compositional analyses indicated that they also contained GalNAc. We have tentatively designated the new disaccharide structure GalNAc beta 1, 4Glc- the "schistocore", which may represent a new type of glycosphingolipid core series.

Animals↗

PubMed1992

The selectin family of carbohydrate-binding proteins: structure and importance of carbohydrate ligands for cell adhesion.

Protein-carbohydrate interactions have been found to be important in many steps in lymphocyte recirculation and inflammatory responses. A family of carbohydrate-binding proteins or lectins, termed selectins, has been discovered and shown to be involved directly in these processes. The three known selectins, termed L-, E- and P-selectins, have domains homologous to other Ca(2+)-dependent (C-type) lectins. L-selectin is expressed constitutively on lymphocytes, E-selectin is expressed by activated endothelial cells, and P-selectin is expressed by activated platelets and endothelial cells. Here, we review the nature of the carbohydrate determinants in tissues recognized by these selectins. The expression of specific sialylated, fucosylated and sulfated carbohydrates in activated endothelium and high endothelial venules promotes interactions with L-selectin on leukocyte surfaces. In contrast, E- and P-selectins recognize specific carbohydrate determinants related to sialyl Le(x) antigen on neutrophil and monocyte surfaces. The discovery of the selectins has generated excitement among glycoconjugate researchers that other carbohydrate-binding proteins and their cognate ligands will be found to function in regulating many types of cellular interactions.

Amino Acid Sequence↗

PubMed1992

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.

Animals↗

PubMed1992

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

Antibodies, Monoclonal↗

PubMed1992

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.

Acetylgalactosamine↗

PubMed1991

Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide.

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.

Animals↗

PubMed1991

The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides.

The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.

Acetylgalactosamine↗

PubMed1991

A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin.

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.

Acetylglucosamine↗

PubMed1991

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing.

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.

Asparagine↗

PubMed1991

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells.

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.

Amino Sugars↗

PubMed1990

LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin.

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.

Animals↗

PubMed1990

The S-type lectin from calf heart tissue binds selectively to the carbohydrate chains of laminin.

We report that the S-type lectin in calf heart tissue, termed calf heart agglutinin (CHA), binds to immobilized mouse laminin in ligand blotting and solid-phase radioligand binding assays. When compared with other glycoproteins, radioiodinated CHA binds preferentially to immobilized laminin. The binding is saturable with a Kd of 9.2 x 10(-7) M and is competitively inhibited by nonradiolabeled CHA as well as a similar lectin from porcine heart tissue. Both lactose and N-acetyllactosamine are good inhibitors of binding to laminin but binding is not inhibited by heparin. Exoglycosidase treatments demonstrated that the binding of radioiodinated CHA to laminin is not dependent on terminal sialyl-, fucosyl-, beta- or alpha-linked galactosyl residues, whereas treatment of laminin with endo-beta-galactosidase significantly decreases the lectin binding. Thus, CHA binds selectively to the poly-N-acetyllactosamine chains on complex-type Asn-linked oligosaccharides in laminin.

Animals↗

PubMed1990

Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA.

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.

Amino Acid Sequence↗

PubMed1990

Transfer and expression of a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase gene in transfected Chinese hamster ovary cells. Competition reactions between the alpha 1,3-galactosyltransferase and the endogenous alpha 2,3-sialyltransferase.

The cDNA encoding a murine UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase has recently been cloned and sequenced using a transient expression method (Larsen, R.D., Rajan, V.P., Ruff, M.M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8227-8231). This report describes the construction and analysis of a Chinese hamster ovary (CHO) cell line in which in vitro expression alpha 1,3-galactosyltransferase activity has been achieved via transfer and expression of the murine alpha 1,3-galactosyltransferase gene. A primary aim of this research was to explore the role of the alpha 1,3-galactosyltransferase in regulating glycoprotein and glycolipid biosynthesis. CHO cells were cotransfected with murine genomic DNA fragments from F9 cells and plasmid DNA containing a resistance gene to the antibiotic G418. Cells resistant to G418 were then selected for expression of surface glycoconjugates containing terminal alpha 1,3-galactosyl residues by isolating cells bound to immobilized Griffonia simplicifolia-I-B4, a lectin which binds to alpha 1,3-galactosyl residues. A positive, stable transfectant clone, designated Clone 3, was obtained and analyzed for expression of the murine of alpha 1,3-galactosyltransferase. Fluorescence-activated cell sorting demonstrated that Clone 3, but not parental, CHO cells bound significant amounts of fluorescein isothiocyanate-labeled G. simplicifolia-I-B4. Southern and Northern blot analyses using the murine alpha 1,3-galactosyltransferase cDNA demonstrated that clone 3, but not parental, CHO cells contain murine alpha 1,3-galactosyltransferase genomic DNA sequences, and express a homologous transcript that comigrates with the authentic 3.6 kilobase alpha 1,3-galactosyltransferase murine mRNA. Enzyme assays confirmed that clone 3, but not parental CHO cells, contained the alpha 1,3-galactosyltransferase activity and that the level of activity is comparable to that found in F9 cells. [3H]Galactose-labeled glycopeptides and glycolipids were obtained from metabolically radiolabeled parental and Clone 3 cells and were analyzed for the presence of terminal alpha 1,3-galactosyl residues. Complex-type, Asn-linked oligosaccharides from both parental and Clone 3 cells contain the repeating disaccharide [3Gal beta 1, 4GlcNAc beta 1]n or poly-N-acetyllactosamine sequences, but only the poly-N-acetyllactosamine chains from clone 3 cells contained the terminal sequence Gal alpha 1,3Gal beta 1,4GlcNAc beta 1-R.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Human transferrin receptor contains O-linked oligosaccharides.

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.

Acetylgalactosamine↗

PubMed1990

Schistosoma mansoni contains a galactosyltransferase activity distinct from that typically found in mammalian cells.

It has been reported previously that some complex-type Asn-linked oligosaccharides contained in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal galactosyl residues. We report here that extracts from S. mansoni adult male and female worms contain a beta 1,4-galactosyltransferase activity that transfers galactose from the donor substrate UDP-galactose to the acceptor substrate N-acetylglucosamine in a beta 1,4-linkage position to form the disaccharide Gal beta 1,4GlcNAc. In this respect the schistosome-derived activity is similar to that commonly found in mammalian tissues. The kinetic properties, however, of the common beta 1,4-galactosyltransferase activity in mammalian tissues are dramatically altered in the presence of the modifier protein alpha-lactalbumin, whereas the beta 1,4-galactosyltransferase activities in adult male and female schistosomes are not altered by this modifier. Overall, our results demonstrate that adult schistosomes contain a beta 1,4-galactosyltransferase activity and that it is unlike that commonly found in mammalian tissues.

Acetylglucosamine↗

PubMed1989

Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells.

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.

Animals↗