Bo E. Samuelsson

Lewis histo-blood group system and associated secretory phenotypes

This review summarises present knowledge of the chemistry, immunology, genetics and clinical significance of antibodies in the Lewis and secretor histoblood group systems. Although red cell serology has laid the foundations for these systems, more recent advances have been made by studying Lewis and related glycoconjugates with monoclonal antibodies, determining structures by mass spectrometry and NMR spectroscopy, identifying enzymes and their specificities, and identifying the genes by molecular biology. The expression of Lewis system antigens is dependent on Lewis and secretor loci. Fucosyltransferases coded by genes at these loci compete and interact with each other and with other transferases to determine an individuals Lewis and secretor phenotype. Exocrine epithelial cells, mostly of endodermal origin, synthesise the Lewis antigens which, as plasma glycolipids, are secondarily acquired by cells of the peripheral circulation. Phenotyping red cells is often regarded as a simple way of determining the Lewis and sometimes the secretor status of an individual; however, the red cell phenotype is influenced by many factors and may not necessarily reflect someones Lewis and secretor genotypes. Two main red cell Lewis groups are usually found, Lewis negative and Lewis positive. In Lewis‐negative individuals, the secretor genotype does not affect the Lewis phenotype, but in Lewis‐positive individuals, the non‐secretor genotype generates the Le(a+b–) phenotype, the secretor genotype causes the Le(a–b+) phenotype, and the partial secretor genotype gives rise to the Le(a+b+) phenotype.

The sphingolipid composition of bovine kidney cortex, medulla and papilla.

Abstract 1. 1. The major sphingolipids of bovine kidney cortex, medulla and papilla were prepared, weighed and characterized. Each of these regions had its own sphingolipid pattern. 2. 2. Sphingomyelins were the major sphingolipid class of bovine kidney. Their concentration decreased from cortex to papilla. 3. 3. Free ceramides were present in considerable amounts. They also decreased in concentration from cortex to papilla. 4. 4. The main glycosphingolipids were glucosylceramides, galactosylceramides and galactosylceramide sulphate esters (sulphatides). Diglycosylceramides and triglycosylceramides were smaller fractions, and tetraglycosylceramides were not detected. Gangliosides were not isolated. 5. 5. Galactosylceramides and sulphatides were concentrated in the medulla, while the cortex and papilla had lower concentrations. 6. 6. The level of glucosylceramides was approximately the same in the cortex as in the medulla, but it was much higher in the papilla. 7. 7. Diglycosylceramides and triglycosylceramides, measured together, increased gradually from cortex to papilla. In the cortex their ratio was about 1: 2, in the medulla 1:1, and in the papilla 3:2. 8. 8. A simple thin-layer Chromatographie study on the phospholipid patterns of the cortex and medulla revealed no differences in relative amounts. 9. 9. The (Na+-K+-ATPase, determined as ouabain-sensitive ATPase, had about the same activity in the cortex as in the medulla.

Lipid pattern and Na+−K+-dependent adenosine triphosphatase activity in the salt gland of duck before and after adaptation to hypertonic saline

SummaryDucks (Anas platyrhynchos) were fed hypertonic saline for eight days, resulting in an activation and hypertrophy of the salt gland. The Na+−K+-dependent adenosine triphosphatase, an enzyme generally assumed to be part of the active Na transport system, increased its specific activity by about 200% during this activation. Sulfatides, the major glycolipids of the salt gland, increased their concentration to the same extent. Cholesterol, cerebrosides, and six phospholipid classes showed an increase of 20–80%.

Limited specificity of xenoantibodies in diabetic patients transplanted with fetal porcine islet cell clusters. Main antibody reactivity against α‐linked galactose‐containing epitopes

Abstract: The immunological specificity of antibodies formed as a result of xenotransplantation of fetal porcine islet‐like cell clusters to diabetic patients was characterized. High titer increases were recorded against porcine cells, solubilized membrane fractions of porcine cells, and purified MHC class I antigens. However, titer increases were also noted against ssDNA and dsDNA and against pig thyroglobulin but not against actin, myoglobin, or haptenated BSA. Antibody titers against tetanus toxoid were unaffected. The reactivity against porcine RBC could be completely blocked by absorption with pig thyroglobulin. Since pig thyroglobulin contains the galαl‐3gal antigen, the reactivity against RBC was most probably mainly due to antibodies against this oligosaccharide epitope. The reactivity against porcine mononuclear cells was only partially absorbed by pig thyroglobulin, indicating a heterogeneity of the clonal response. These conclusions were substantiated by data showing that the antigenic determinants on pig thyroglobulin were completely destroyed by treatment with α‐ but not with β‐galactosidase. Further studies showed that immune reactivity against pig RBCs, platelets, islet‐cells, endothelial cells and pig MHC class I molecules, caused by xenoimmunization, was almost completely blocked by either absorption of antibodies on a column of Sepharose beads coated with galα1‐3gal or by pretreatment of the antigen fractions with α‐galactosidase. Western blot experiments revealed that both the natural and xenoimmune antibodies reacted with a large number of different glycoproteins. There was no difference in heterogeneity of the response when comparisons were made between pre‐and posttransplantation sera, nor was there any difference of patterns caused by IgM or IgG antibodies. Absorption studies revealed that this epitope was present in a large number of different glycoproteins, a conclusion verified by staining with the eluate from a thyroglobulin‐immunosorbent column or with the eluate from the galα1‐3gal‐coupled Sepharose column. Our findings demonstrate that the xenoimmune response is mainly directed against oligosaccharide residues and that there is a limited clonal heterogeneity of antibodies in xenografted patients. There was no direct evidence of reactivity against any porcine proteins nor of specific immunization against porcine MHC peptides. The data form a basis for a future understanding of means to cope with and prevent the rejection of xenogeneic islet cells in man. We also suggest, based on the specificity found in xenoimmunized patients sera, that a major part of naturally occurring antibodies might be specifically reactive with carbohydrate antigens.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: The globo-series (one to five sugars)

Abstract Proton nuclear magnetic resonance spectroscopy has been reevaluated concerning the assignment of anomeric structure of glycosphingolipids. Solubility problems due to a varying number of sugars are avoided by permethylation, allowing a wide range of glycolipids to be compared. High resolution spectra were recorded in chloroform solution for the following substances with known structure, most of them representing a successive building up of members of the globo-series: ceramide, Galβ1 → 1Cer, a mixture of Glcα1 → 1Cer and Glcβ1 → 1Cer, lactosylceramide, globotriaosylceramide, globotetraosylceramide (globoside), and GalNAcα1 → 3globotetraosylceramide (Forssman hapten). Resonances originating in anomeric protons were identified and possible interference from other signals was defined. A complex set of resonances from H-1 of hexosamines was probably due to two separate conformers of the acetamido group caused by N -methylation. The complexity disappeared upon reduction with LiAlH 4 . The chemical shifts and coupling constants were characteristic for the configuration of the glycosidic bond, the type of monomer, and in part for its location in the chain. At present, spectra may be recorded from 200-μg samples. It is concluded that the good quality and resolution obtained make this technique an alternative method to the presently used enzymatic degradation for establishing anomeric structure of glycosphingolipids.

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.The results suggest that glycolipids of erythrocytes and possibly of other cell types bear the A type 3/4 determinant specific for the A1 subgroup and that A type 3/4 determinants of glycoproteins might be present in both A1 and A2 subgroups on short oligosaccharide chains which are only detectable at the level of the Golgi apparatus.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Lewis-active and Lewis-like substances.

Abstract High resolution nuclear magnetic resonance spectra were recorded in a chloroform solution of six Lewis-active or Lewis-like glycosphingolipids in permethylated and permethylated-reduced (LiAlH4) form. The samples were selected to cover the presently known structural variants of α-fucose linked to galactose and N-acetylglucosamine. Fucα1 → 2Gal, Fucα1 → 3GlcNAc, and Fucα1 → 4GlcNAc gave characteristic and well-separated anomeric resonances. Furthermore, upon reduction there was a strong deshielding effect on Fucα1 → 3GlcNAc and Galβ1 → 3GlcNAc (linkage vicinal to reduced amide), which makes it possible to differentiate type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains. This improved method of nuclear magnetic resonance spectroscopy is discussed in relation to sequence analysis by mass spectrometry, two microscale structural methods using the same type of derivatives and needing no degradations before analysis.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: Blood group ABH-active substances

Abstract High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH 4 ) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.

Separation of monoglycosylceramides (cerebrosides) of bovine kidney into subgroups and characterization by mass spectrometry

Monoglycosylceramides of bovine kidney have been separated on columns of borate-impregnated silica gel G into six fractions homogeneous on thin-layer plates. The lipophilic components were identified by mass spectrometry of trimethylsilyl ethers of intact lipids, and the carbohydrate identified by gas chromatography after degradation. Dihydroxy and trihydroxy long-chain bases, normal and 2-hydroxy fatty acids, and glucose and galactose were the components of eight possible combinations. Two of these made up 90 % of the total weight, namely glucosylceramide with trihydroxy base and hydroxy fatty acid, and galactosylceramide with dihydroxy base and hydroxy fatty acid.

Mass spectrometry of mixtures of intact glycosphingolipids

Publisher Summary This chapter discusses mass spectrometry of mixtures of intact Glycosphingolipids. Glycosphingolipids usually occur in nature in complex mixtures and in small amounts. The number of sugars may range from one to about forty. These substances show distinct cell, tissue, strain, and species specificity in their structures and undergo successive changes during development and on tumor transformation. Of particular biomedical importance are the determinants within the major blood group systems (ABO, Lewis, I, P) and the glycolipids which may act as tumor-associated antigens and as receptors for microbes. Structural studies are, therefore, of major importance for the progress of transplantation and tumor biology and for the understanding of infectious diseases. Such studies require sensitive and rapid methods giving information with high structural specificity and with the capability to work with mixtures. Mass spectrometry is not the only method that matches these demands, but taken overall it is perhaps the most valuable tool in this respect. The electron ionization (EI)/MS distillation technique for the analysis of glycosphingolipid mixtures is the principal subject of this chapter. A few aspects of TLC/MS are also included.