Biomedical subjects

Howard R Morris

Publications and source records attributed to Howard R Morris.

45 records · Page 3Linked to original sources

PubMed2002

The expression of free oligosaccharides in human seminal plasma.

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.

Carbohydrate Sequence↗

PubMed2002

Characterization of a putative alpha-mannosyltransferase involved in phosphatidylinositol trimannoside biosynthesis in Mycobacterium tuberculosis.

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an alpha-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625-31631], which catalysed the formation of triacyl (Ac(3))-PIM(2) (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[(14)C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac(3)PIM(3) (i.e. the trimannoside). The results indicate that pimC encodes an alpha-mannosyltransferase involved in Ac(3)PIM(3) biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette-Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.

Amino Acid Sequence↗

PubMed2002

Differences between neonates and adults in carbohydrate sequences and reaction kinetics of plasmin and alpha(2)-antiplasmin.

This study investigates reaction kinetics by slow-binding kinetics methods of both adult and fetal plasmin (Types 1 and 2) with adult and fetal alpha(2)-antiplasmin. In addition, carbohydrate sequences of Fetal and Adult Plasminogen Types 1 and 2, as well as fetal and adult alpha(2)-antiplasmin, were determined by mass spectrometric analysis. All curves of plasmin-alpha(2)-antiplasmin interaction followed the same pattern, indicating reversible slow-binding inhibition with an initial loose complex and a following tight complex. Differences between fetal and adult plasmin reactions with alpha(2)-antiplasmin were predominantly due to the initial loose complex. Values for K(i initial) in the reaction with adult alpha(2)-antiplasmin were 1.5 and 1.6 nM for Fetal Plasmin Types 1 and 2, respectively; compared to 0.3 and 0.7 nM for the corresponding adult types. Increasing concentrations of tranexamic acid resulted in a continuous increase of K(i initial) until a plateau was reached which was similar for all plasmin types. Almost identical values could be obtained when fetal alpha(2)-antiplasmin was used instead of adult alpha(2)-antiplasmin. Mass spectrometric analyses of the glycans present on plasminogen revealed a higher level of truncated N-glycans on the fetal material compared to the adult. The O-glycans of fetal and adult plasminogen were closely similar and only minor differences were observed between N-glycans of fetal and adult alpha(2)-antiplasmin. In conclusion, both fetal plasmin isoforms are less inhibited by alpha(2)-antiplasmin compared to the adult plasmin variants. These findings are important for the understanding of the physiology of the fibrinolytic system in neonates and provide further evidence that differences in glycosylation could be associated with marked effects on protein function.

Adult↗

PubMed2002

A putative serine protease among the excretory-secretory glycoproteins of L1 Trichinella spiralis.

Trichinella spiralis first-stage larvae infect susceptible hosts by invading epithelial cells that line the small intestine. During this process the larva disgorges several glycoproteins that bear an unusual, highly antigenic sugar moiety, tyvelose (3,6-dideoxy arabinohexose). Monoclonal antibodies specific for tyvelose protect the intestine against infection, implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment in the intestinal epithelium. In order to investigate these glycoproteins at the molecular level, we first prepared monoclonal anti-peptide antibodies. The antibodies bind a family of glycoproteins that are present in excretory-secretory products of first-stage larvae and are delivered to epithelial cells during invasion by T. spiralis. The major species present in an affinity purified fraction of crude T. spiralis antigens were subjected to tryptic peptide digestion. De novo amino acid sequencing of the peptides using Q-TOF tandem mass spectrometry, in combination with database searches and antibody screening of an L1 cDNA library, showed that the glycoproteins are variably glycosylated homologues of the serine protease family.

Amino Acid Sequence↗

PubMed2002

In vivo glycosylation of MUC1 in airway epithelial cells.

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.

Caco-2 Cells↗

PubMed2002

MS screening strategies: investigating the glycomes of knockout and myodystrophic mice and leukodystrophic human brains.

The implementation of highly sensitive and rapid mass spectrometric screening strategies for defining the glycosylation repertoires of organs in knockout mice is helping to reveal the roles that glycans play in health and disease. Thus novel glycosylation pathways have been uncovered in two such knockouts, namely alpha-mannosidase II null mice and UDP-N-acetylglucosamine: alpha 6-D-mannoside beta 1,2-N-acetylglucosaminyltransferase II null mice. This chapter documents the glycosylation profiles of a wide range of organs from the normal mouse which should facilitate future glycomics studies of knockout mice. Furthermore, we report applications of our screening technology in studies of the myodystrophy mouse and a human leukodystrophy.

Animals↗

PubMed2002

Structural characterization of lipo-oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system.

The two-component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhimurium. In Yersinia pestis, which expresses lipo-oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild-type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild-type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild-type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.

Bacterial Proteins↗

PubMed2002

Identification of major outer surface proteins of Streptococcus agalactiae.

To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.

Amino Acid Sequence↗

PubMed2002

The glycomes of Caenorhabditis elegans and other model organisms.

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fuc alpha 1-2 Gal 1-2 Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

Amidohydrolases↗