Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans

The core 1 beta1-3-galactosyltransferase (T-synthase) transfers Gal from UDP-Gal to GalNAcalpha1-Ser/Thr (Tn antigen) to form the core 1 O-glycan Galbeta1-3GalNAcalpha1-Ser/Thr (T antigen). The T antigen is a precursor for extended and branched O-glycans of largely unknown function. We found that wild-type mice expressed the NeuAcalpha2-3Galbeta1-3GalNAcalpha1-Ser/Thr primarily in endothelial, hematopoietic, and epithelial cells during development. Gene-targeted mice lacking T-synthase instead expressed the nonsialylated Tn antigen in these cells and developed brain hemorrhage that was uniformly fatal by embryonic day 14. T-synthase-deficient brains formed a chaotic microvascular network with distorted capillary lumens and defective association of endothelial cells with pericytes and extracellular matrix. These data reveal an unexpected requirement for core 1-derived O-glycans during angiogenesis.     

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.     

Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc and might require invertebrate-specific factors for the formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and open new avenues to explore O-glycan function in this model organism.     

Cloning and expression of human core 1 beta1,3-galactosyltransferase.

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T., Cummings, R. D., and Canfield, W. M. (2002) J. Biol. Chem. 277, 169-177) were used to identify the core 1 beta3-Gal-T sequences in the human expressed sequence tag data bases. A 1794-bp human core 1 beta3-Gal-T cDNA sequence was determined by sequencing the expressed sequence tag and performing 5'-rapid amplification of cDNA ends. The core 1 beta3-Gal-T predicts a 363-amino acid type II transmembrane protein. Expression of both the full-length and epitope-tagged soluble forms of the putative enzyme in human 293T cells generated core 1 beta3-Gal-T activity that transferred galactose from UDP-Gal to GalNAcalpha1-O-phenyl, and a synthetic glycopeptide with Thr-linked GalNAc and the product was shown to have the core 1 structure. Northern analysis demonstrated widespread expression of core 1 beta3-Gal-T in tissues with a predominance in kidney, heart, placenta, and liver. Highly homologous cDNAs were identified and cloned from rat, mouse, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that the enzyme is widely distributed in metazoans. The core 1 beta3-Gal-T sequence has minimal homology with conserved sequences found in previously described beta3-galactosyltransferases, suggesting this enzyme is only distantly related to the known beta3-galactosyltransferase family.     

Cosmc和T-合酶在黏蛋白型O-聚糖合成中的重要作用及其与人类疾病的相关性

从核心1结构（Galβ1,3GalNAcα1-O-Ser/Thr, core 1 structure, T antigen）中衍生出来的黏蛋白型O-聚糖在很多生理过程中发挥重要的生物学功能。T-合酶 （core 1 β3-galactosyltransferase, T-synthase） 是合成核心1结构的唯一糖基转移酶,它主要的功能是将半乳糖（Galactose） 添加到GalNAcα1-Ser/Thr （Tn抗原） 糖链上。但是在人体和其他脊椎动物中有活性的T-合酶的形成需要一个重要的伴侣分子Cosmc;Cosmc功能丧失将直接导致T-合酶失活,其结果是机体细胞只能合成Tn抗原以及唾液酰化Tn （sialylTn, STn, Neu5Acα2,6GalNAcα1-O-Ser/Thr）。综述目前对T-合酶和Cosmc的研究以及他们在人类疾病（如异常O-聚糖表达相关的Tn综合征、IgA肾病和肿瘤）发生发展中的作用。     

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.     

Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.     

Differential contributions of recognition factors of two plant lectins -Amaranthus caudatus lectin and Arachis hypogea agglutinin, reacting with Thomsen-Friedenreich disaccharide (Galbeta1-3GalNAcalpha1-Ser/Thr)

Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for the Thomsen-Friedenreich (T(alpha), Galbeta1-3GalNAcalpha1-Ser/Thr) glycotope, but differ in monosaccharide binding--GalNAc>Gal (inactive) for ACL; Gal>GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was based on studies with a small number monosaccharides and T-related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that the main recognition factors of ACL, GalNAc was the only monosaccharide recognized by ACL as such, its polyvalent forms (poly GalNAcalpha1-Ser/Thr, Tn in asialo OSM) were not recognized much better. Human blood group precursor disaccharides Galbeta1-3/4GlcNAcbeta (I(beta)/II(beta)) were weak ligands, while their clusters (multiantennary II(beta)) and polyvalent forms were active. The major recognition factors of PNA were a combination of alpha or beta anomers of T disaccharide and their polyvalent complexes. Although I(beta)/II(beta) were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galbeta1-3GalNAc (T), while the PNA binding domain is proposed to be Galbeta1-3GalNAcalpha or beta1--as the major combining site with an adjoining subsite (partial cavity type) for a disaccharide, and most complementary to the linear tetrasaccharide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glc (T(beta)1-4L, asialo GM(1) sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes for the interaction with these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions.     

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.     

The function of PS integrins during Drosophila embryogenesis

The Drosophila position-specific (PS) antigens are homologous to the vertebrate fibronectin receptor family, or integrins. A Drosophila gene required for embryonic morphogenesis, l(1)myospheroid, codes for a product homologous to the beta subunit of the vertebrate integrins. l(1)myospheroid mutants die during embryogenesis. We show here that they lack the beta subunit of the PS antigens. In the absence of the beta subunit in mutant embryos, the PS alpha subunits are not expressed on the cell surface. We conclude that the l(1)myospheroid phenotype represents the lack-of-function phenotype for these Drosophila integrins. In wild-type embryos, PS antigens are found at the interface between mesoderm and ectoderm, and later mainly at the attachment sites of muscles to the epidermis and gut. Together these results indicate that during embryogenesis, Drosophila integrins are used to attach mesoderm to ectoderm, and are required for the proper assembly of the extracellular matrix and for muscle attachment.     

Beta 1,4-galactosyltransferase (beta 4GalT)-IV is specific for GlcNAc 6-O-sulfate. Beta 4GalT-IV acts on keratan sulfate-related glycans and a precursor glycan of 6-sulfosialyl-Lewis X

The Galbeta1-->4(SO(3)(-)-->6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta1,4-galactosyltransferase (beta4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta4GalT has a similar sequence to human beta4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta1-->3(SO(3)(-)-->6GlcNAcbeta1-->6) GalNAcalpha1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta4GalT-IV is a 6SGN-specific beta4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.     

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.