Pamela Greenwell

Blood group antigens: molecules seeking a function?

The blood group antigens have been dismissed by some researchers as merely ‘icing on the cake’ of glycoprotein structures. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. This paper reviews the research which suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer. It explores the evidence and poses questions as to the relevance and implications of the results.

Regulation of expression of carbohydrate blood group antigens.

The carbohydrate antigens associated with the human ABO and Lewis blood group systems are excellent models for the study of the genetic regulation of glycoconjugate biosynthesis because their expression on erythrocytes and in saliva has been thoroughly investigated in terms of classical genetics and the chemical structures and pathways for the formation of the antigens are now well understood. The primary protein products of the blood group genes are believed to be the glycosyltransferase enzymes that complete the biosynthesis of the determinants. The important controlling factors still to be elucidated are the genetic and environmental influences leading to the tissue specific expression of these antigens. The 3 types of regulation mechanisms discussed in this review are those arising: 1) from the specificity requirements of the glycosyltransferases encoded by the blood group genes; 2) from the competition or co-operation of glycosyltransferases encoded by genes at the same or independent loci; and 3) from the existence and tissue distribution of glycosyltransferases with related, but not identical, substrate specificities.

UDP-N-acetyl-D-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene at the human blood group ABO locus

The properties of the enzyme in the serum of blood group B individuals that catalyses the transfer of small amounts of N-acetyl-D-galactosamine to H-active precursor structures were compared with those of the blood group B gene-associated alpha-(1----3)-D-galactosyltransferase and with the blood group A gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases in the serum of blood group A1 and A2 individuals. The biosynthetic products formed by the enzyme in B serum were identical with the A-active structures synthesised by the A1 and A2 gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases but the enzyme differed from the A1 and A2 transferases in its apparent Km for UDP-N-acetyl-D-galactosamine, its heat susceptibility, its failure to bind to Sepharose 4B, and its adsorption to H-active sites on group O red cell ghosts under conditions which bind the B transferase but fail to adsorb the A1 and A2 transferases. The correlation between the levels of alpha-(1----3)-D-galactosyltransferase and alpha-(1----3)-N-acetyl-D-galactosaminyltransferase activities in all the group B serum samples tested, the maintenance of the same ratio of activities after successive cycles of binding to group O red cell ghosts, the retention of the ability to convert blood group O to A-active cells after treatment of the serum with Sepharose 4B, and the failure to detect any comparable activity in group O serum samples tested under the same conditions indicated that the enzyme in group B serum that utilises UDP-N-acetyl-D-galactosamine to make blood group A-active structures is the B gene-associated alpha-(1----3)-D-galactosyltransferase.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer.

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

Buttressing volatile desktop grids with cloud resources within a reconfigurable environment service for workflow orchestration

Cloud technology has the potential for widening access to high-performance computational resources for e-science research, but barriers to engagement with the technology remain high for many scientists. Workflows help overcome barriers by hiding details of underlying computational infrastructure and are portable between various platforms including cloud; they are also increasingly accepted within e-science research communities. Issues arising from the range of workflow systems available and the complexity of workflow development have been addressed by focusing on workflow interoperability, and providing customised support for different science communities. However, the deployments of such environments can be challenging, even where user requirements are comparatively modest. RESWO (Reconfigurable Environment Service for Workflow Orchestration) is a virtual platform-as-a-service cloud model that allows leaner customised environments to be assembled and deployed within a cloud. Suitable distributed computation resources are not always easily affordable and can present a further barrier to engagement by scientists. Desktop grids that use the spare CPU cycles available within an organisation are an attractively inexpensive type of infrastructure for many, and have been effectively virtualised as a cloud-based resource. However, hosts in this environment are volatile: leading to the tail problem, where some tasks become randomly delayed, affecting overall performance. To solve this problem, new algorithms have been developed to implement a cloudbursting scheduler in which durable cloud-based CPU resources may execute replicas of jobs that have become delayed. This paper describes experiences in the development of a RESWO instance in which a desktop grid is buttressed with CPU resources in the cloud to support the aspirations of bioscience researchers. A core component of the architecture, the cloudbursting scheduler, implements an algorithm to perform late job detection, cloud resource management and job monitoring. The experimental results obtained demonstrate significant performance improvements and benefits illustrated by use cases in bioscience research.

THE N-GLYCANS OF TRICHOMONAS VAGINALIS CONTAIN VARIABLE CORE AND ANTENNAL MODIFICATIONS

Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data have been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and high-performance liquid chromatography data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.

Glycosidases in mucin-dwelling protozoans.

A range of protozoans were tested for the presence of glycosidases using p-nitrophenyl sugars as substrates. Some of the organisms were mucin dwellers whereas others were blood borne parasites. It had been hypothesized that glycosidase production would be significantly higher in the mucin dwellers. The results obtained demonstrated that the urogenital protozoans Tritrichomonas foetus and Trichomonas vaginalis produced a vast range of glycosidases which included those required for mucin breakdown. The gut dwelling protozoans Giardia lamblia and Entamoeba histolytica both produced β-N-acetylglucosaminidase. G.lamblia also had detectable β N-acetylgalactosaminidase activity, and small amounts of β mannosidase were found in the extracts from E. histolytica. In contrast, little or no glycosidase activity was detected under the same experimental conditions in Leishmania donovani, Trypanosoma brucei or T. cruzi. The mucin dwelling protozoans all produce β-N-acetylglucosaminidase but only the Trichomonads produced the range of enzymes required for complete breakdown of mucin. This seems to suggest that mucin breakdown is not a characteristic of all mucin dwelling protozoans.

Trichomonas vaginalis: paradigm of a successful sexually transmitted organism

Abstract Trichomonas vaginalis (TV) is one of the most successful protozoan pathogens and one of the most common sexually transmitted organism in females, yet it is also one of the most poorly investigated. By producing a wide array of glycosidases and cysteine proteinase enzymes, the organism can easily adapt to the environment, harvesting host proteins and DNA for metabolism. With the ability to cause lesions, vaginitis and acute inflammatory disease of the genital mucosa, TV acts as a potential catalyst in the acquisition of secondary infections including human immunodeficiency virus (HIV) and human papillomavirus (HPV), the organism responsible for the pathogenesis of cervical cancer. Treatment of TV infection is relatively easy and could dramatically reduce the transmission of HIV in areas where TV is endemic.

Fucosyltransferase activities in human lymphocytes and granulocytes: Blood group H-gene-specified α-2-L-fucosyltransferase is a discriminatory marker of peripheral blood lymphocytes

Human lymphocytes and granulocytes were examined for blood group H‐gene‐specified α‐2‐L‐fucosyl‐transferase, α‐3‐L‐fucosyltransferase and blood group Le‐gene‐specified α‐4‐L‐fucosyltransferase. No α‐4‐L‐fucosyltransferase was detectable in either cell type. α‐3‐L‐Fucosyltransferase was readily demonstrable in both lymphocytes and granulocytes and the levels of activity per 106 cells were approximately the same. In contrast, α‐2L‐fucosyltransferase was detectable in lymphocytes but not in similar numbers of granulocytes. The absence of α‐2‐L‐fucosyltransferase was confirmed in granulocytes isolated from the blood of eighty unselected donors and was independent of ABO blood group or ABH secretor status.

Parameter Sweep Workflows for Modelling Carbohydrate Recognition

Carbohydrate recognition is a phenomenon critical to a number of biological functions in humans. Understanding the dynamic behaviour of oligosaccharides should help in the discovery of the mechanisms which lead to specific and selective recognition of carbohydrates by proteins. Computer programs which can provide insight into such biological recognition processes have significant potential to contribute to biomedical research if the results of the simulation can prove consistent with the outcome of conventional wet laboratory experiments. In order to validate these simulation tools and support their wider uptake by the bio-scientist research community, high-level easy to use integrated environments are required to run massively parallel simulation workflows. This paper describes how the ProSim Science Gateway, based on the WS-PGRADE Grid portal, has been created to execute and visualise the results of complex parameter sweep workflows for modelling carbohydrate recognition.