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Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4-8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O-acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.

Summary: The open access comprehensive GlycoCD database application is for representation and retrieval of carbohydrate-related clusters of differentiation (CDs). The main objective of this database platform is to provide information about interactions of carbohydrate moieties with proteins that are important for identification of specific cell surface molecule with a focus on the integration of data from carbohydrate microarray databases. GlycoCD database comprises two sections: the carbohydrate recognition CD and glycan CD. It allows easy access through a user-friendly web interface to all carbohydrate-defined CDs and those that interact with carbohydrates along with other relevant information. Availability: The database is freely available at http://glycosciences.de/glycocd/index.php Contact: r.s-albiez@dkfz.de

aThe Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, United Kingdom; bCentre d'Immunologie, Parc Scientifique de Luminy, Cedex, France; cFaculte de Medicine de Creteil, Cedex, France; dDivision of Immunity and Infection, MRC Center for Immune Regulation, Department of Rheumatology, University of Birmingham, Edgbaston, Birmingham, United Kingdom; eJohns Hopkins Comprehensive Cancer Center, Baltimore, Maryland, USA; fDepartment of Microbiology, University of Washington, Seattle, Washington, USA; gCLB, Department of Experimental Immunohematology, Amsterdam, The Netherlands; hLaboratory of Molecular Hematology/Division of Molecular Medicine, North Shore University Hospital, Manhasset, New York, USA; iKinderklinik, Medizinische Hochschule Hannover, Hannover, Germany; jMater Medical Research Institute, Aubigny Place, Mater Hospital, Queensland, Australia; kInstitute of Molecular Genetics AS CR, Prague, Czechoslovakia; lInstitut fur Immunologie der Univ. Heidelberg, Heidelberg, Germany, mProfessor of Biochemistry, University of Illinois College of Medicine, Urbana, Illinois, USA; nGerman Cancer Research Center, Heidelberg, Germany; oNational Institute of Health, Bethesda, Maryland, USA; pDirector of Biology Celltech RD qInstitute for Research in Biomedicine, Bellinzona, Switzerland; rUniversity of Adelaide, Department of Pediatrics, Women's and Children's Hospital, South Australia, Australia

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of “blind” antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

The most recent Human Leucocyte Differentiation Antigen Workshop ("HLDA7") took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid "blind" screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new "intracellular CD" categorisation scheme is devised for such molecules).

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.

We found that human peripheral B and T cells differed in the surface expression of alpha2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galss1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the alpha2-6 sialylated model gangliosides IV6NeuAcnLc4Cer (2-6 SPG), VI6NeuAcnLc6Cer (2-6 SnHC), VIII6NeuAcnLc8Cer (2-6 SnOC), and X6NeuAcnLc10Cer (2-6 SnDC). We found that, in addition to their common requirement of an alpha2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar alpha2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

SUMMARYAge-related changes in functional subsets of lymphocytes may influence the potential to build up immune responses. In particular, the capacity of tonsillar lymphocytes to counter infections may be altered during ageing. In order to address this question we investigated the proportional distribution of several subsets of tonsillar T and B cells with regard to ageing. Tonsils were derived from 119 patients between 2 and 65 years of age. Lymphocyte subsets were monitored by three-colour fluorescence of relevant CD markers in flow cytometry. As a general tendency the percentage of CD3+ T cells steadily increased whereas that of CD19+ B cells decreased at the same time. No significant differences were observed between lymphocytes of patients with and without inflammatory history of the tonsils. The percentage of CD8+ T cells declined whereas that of CD4+ T cells increased during the same time span. CD45RA+ T cells increased during the first two decades of life and gradually decreased thereafter. In contrast, CD45RO+ T cells showed an opposite trend. No differences were seen in the population of CD3−/CD56+ natural killer (NK) cells. The mature B cell marker CD40 showed no significant changes during ageing. However, CD38+ B cells, representing B cells of late maturation stages, dramatically declined up to the age of 65. In a similar manner the CD5+ subpopulation of B cells decreased during ageing. Substantial changes in major tonsillar T and B cell populations as shown in this study may have an impact on the ageing process of the immune system.