Bo Samuelsson

Molecular basis for erythrocyte Le(a+ b+) and salivary ABH partial-secretor phenotypes: expression of a FUT2 secretor allele with an A-->T mutation at nucleotide 385 correlates with reduced alpha(1,2) fucosyltransferase activity.

TheSewA385T mutation of the FUT2 gene was found to correlate with both the erthrocyte Le(a+b+) and/or salivary ABH partial-secretor phenotypes of Polynesians. Constructs with FUT1 and FUT2 wild type genes, and the FUT2SewA385T,seG428A andseC571T mutated alleles, were cloned into pcDNAI, and expressed in COS-7 cells. COS-7 cells transfected with theSewA385T allele had weak, but detectable, α(1,2)fucosyltransferase activity, with an acceptor substrate pattern similar to the wild type FUT2 gene. Comparative kinetic studies from cell extracts with mutatedSewA385T and wild type FUT2 alleles gave similarKm values, but less enzyme activity was present in cells transfected withSewA385T (Vmax 230 pmol h−1 mg−1), as compared to those transfected with FUT2 (Vmax 1030 pmol h−1 mg−1), suggesting that the mutated enzyme is more unstable. These results confirm that the molecular basis for the erythrocyte Le(a+b+) and the associated ABH salivary partial-secretor phenotype, is an amino acid change of Ile 129→Phe in the secretor α(1,2)fucosyltransferase.

A Second Nonsecretor Allele of the Blood Group α(1,2)Fucosyl‐transferase Gene (FUT2)

While screening Le(a+b+) Polynesian DN A samples for a candidate Sew allele, a point mutation (C571→T) resulting in a new stop codon (Arg191→stop) in the α(1,2)fucosyltransferase gene (FUT2) was identified. This point mutation resulted in the gaining of a new restriction enzyme cleavage site (DdeI), which allowed restriction enzyme cleavage screening of 40 selected Polynesians and 42 random Caucasians. The nonsecretor phenotype in two of the three nonsecretor Polynesians analyzed was due to homozygosity for the ‘new’ mutation, whereas the third Polynesian nonsecretor (with Caucasian ancestors) was due to homozygosity of the ‘old’ (Trp143→stop) mutation. The nonsecretor phenotype in all Caucasians analyzed was a consequence of homozygosity for the ‘old’ mutation. Both the new and the old nonsecretor mutations were identified in the heterozygous state in some secretor‐positive Polynesians, while only the old mutation was found in the heterozygous state in Caucasians of the same phenotype.

Extracorporeal (“ex vivo”) connection of pig kidneys to humans. I. Clinical data and studies of platelet destruction

Abstract: The pioneering experiment by Welsh et al. (Immunological Lett 1991:29:167–170) connecting a pig kidney to the human circulation has been repeated in a modified manner. Two volunteer dialysis patients were pretreated by daily plasmapheresis on days ‐2,‐1, and 0 to remove the naturally occurring anti‐pig xenoantibodies. The anti‐pig lymphocytotoxic liters were reduced from 1:8 to 1:2 in patient 1 and from 1:8 to 1:1 in patient 2. No steroids or immunosuppressive drugs were administrated before or during the experiments. A sterile pig kidney was extracorporeally (“ex vivo”) connected to the patients a/v fistula using an arterial and a venous pump similar to a dialysis. The two experiments gave different results. In the first experiment the perfusion pressure was kept at 100 mmHg for the initial 25 min by reducing the pump speed until the minimum blood flow of 30 ml/min was reached. Thereafter, the pressure rose continuously and the experiment was terminated at 65 min at a perfusion pressure of 200 mmHg. The patient did not feel any discomfort during the perfusion. In the second experiment, a stable blood flow of 200 ml/min was reached at a pressure of 100 mmHg after a few minutes. The perfusion was terminated at 15 min when the patient developed chest and abdominal pain, hypotension, and electrocardiographic signs of myocardial ischemia. The patient recovered quickly. In the first experiment, small volumes of clear urine was produced until the pressure rose above 100 mmHg, which resulted in hematuria. In the second experiment clear urine (4 ml/min) was produced. 51Chromium clearance values were after 15 min <1 ml/min for kidney 1 and 12 ml/min (8 ml/min/100 g) for kidney 2. A drastic reduction in platelet count (128 to 48 and 64 to 8 × 109/1, respectively) during the passage through the kidney was found in blood samples collected simultaneously before and after the organ. No change in hemoglobin values and leucocyte counts were found. Light‐ and electron‐microscopical analysis of the kidney tissues revealed for kidney 1 focal areas with obliteration of the glomerular and peritubular capillaries by platelets and PMN cells and severe damage of the endothelial cells comparable to a picture of a hyperacute rejection. In kidney 2, all vessels were patent but in the capillaries large amount of membrane fragments were detected by electron microscopy and a discrete damage of the endothelial cells were seen in some segments. No intact platelets were present in the vascular tree. These human experiments support the hypothesis that hyperacute rejection of pig to human xenografts is delayed in time by removal of the preformed anti‐pig xenoantibodies. A new finding was a very rapid destruction of platelets occurring in the kidney of patient 2 who had very low liters of xenoantibodies. The humoral immune response is described in detail in an accompanying paper (Rydberg et al., this issue).

Specificities of human IgM and IgG anticarbohydrate xenoantibodies found in the sera of diabetic patients grafted with fetal pig islets

Abstract: Serum samples were collected from four diabetic patients who had received intraportal injections of pig fetal islet‐like cell clusters (ICC). The binding of IgM and IgG antibodies to glycosphingolipid antigens prepared from different pig organs and separated on thin layer plates was investigated in pre‐ and posttransplant serum samples. Both IgM and IgG antibodies in the pretransplant serum samples bind to several glycolipid fractions from the tri‐, tetra‐ and pentasaccharide regions but also to compounds with longer carbohydrate chains. In the posttransplant serum samples stronger binding, compared to the pretransplant samples, was noted for both Ig‐classes. Strong binding was seen in the pentasaccharide region known to contain the “linear blood group B” structure Galαl‐3Galβl‐4GlcNAcβl‐3Galβl‐4Glcβl‐lCer. There was no convincing evidence of the I recognition of new specificities. Adsorption of a patient serum to a Synsorbcolumn with Galαl‐3Gal specificity did not grossly change the binding pattern of the IgG antibodies in the effluate. However, the eluate from the column showed strong binding to the “linear B” compound but also to glycolipids with longer carbohydrate chains presumably with the same terminal epitope. Identification of these are in progress.

Extracorporeal ("ex vivo") connection of pig kidneys to humans. II. The anti-pig antibody response.

Abstract: Pig kidneys were extracorporeally “ex vivo” connected to the circulation of two volunteer male dialysis patients (Breimer et al., this issue). The patients were pretreated by daily plasmapheresis for 3 consecutive days, which reduced the anti‐pig lymphocytotoxic titer from 8 to 2 in the first patient and from 8 to 1 in the second patient. The anti‐pig hemagglutinating titers were reduced from 32 to 4 in the first patient and from 2 to 1 in the second patient. No drugs, except heparin, were given. The perfusion lasted for 65 min in patient 1 and the experiment was terminated due to increased vascular resistance in the pig kidney. Ultrastructural investigation showed a picture similar to a hyperacute vascular rejection. Immunohistochemical studies showed a weak staining of IgM antibodies, but no IgG in the small arteries and glomeruli.

Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry

Extracellular superoxide dismutase, EC-SOD, the main superoxide dismutase in biological fluids, is known from its lectin binding to be a glycoprotein. We have characterized the glycosylation of recombinant EC-SOD. A tryptic digest of the protein contained only one glycosylated peptide. This peptide was specifically bound to lectins and stained by periodic acid-Schiff stain. Although appearing very large on size-exclusion chromatography, it was shown to be glycosylated at only one site, asparagine-89, by specific cleavage with glycanases followed by mass spectrometry of the resulting peptide. Based on the binding properties of the peptide to concanavalin A and lentil lectin and the elution profile of N-glycanase-treated glycopeptide on ion-exchange chromatography, the carbohydrate appears to be the complex biantennary type with a core fucose.

Plasma and Red‐Cell Glycolipid Patterns of Le(a+b+) and Le(a+b‐) Polynesians as Further Evidence of the Weak Secretor Gene Sew

Monoclonal antibodies and thin‐layer chromatography were used to study the unusual erythrocyte Lewis phenotypes found in healthy Polynesians. A single monoclonal anti‐Leb reagent 073 (clone LM129) was found which could detect Leb antigen on the Polynesian erythrocytes of samples that were unreactive with various polyclonal and monoclonal anti‐Leb reagents. Glycolipid fractions prepared from the plasma and erythrocytes of selected Polynesian samples of red‐cell Le(a‐b‐), Le(a+b‐) and Le(a+b+) phenotypes were found to have Leb glycolipids. The Leb antigen in some individuals is so weakly expressed that it is undetectable by routine erythrocyte phenotyping. Unusually large glycolipids bearing the Leb epitope were also found in some Polynesian samples, although the contribution of these novel glycolipids to phenotyping is unclear. The inability to detect Leb by routine methods and the presence of novel structures can be partially explained in terms of the presence of a weak secretor gene Sew.

Structural and immunochemical identification of Lea, Leb, H type 1, and related glycolipids in small intestinal mucosa of a group O Le(a-b-) nonsecretor

Total nonacid glycosphingolipids were isolated from small intestine mucosal scrapings of a red cell blood group O Le(a-b-) nonsecretor cadaver. Glycolipids were extracted and fractionated into five fractions based on chromatographic and immunostaining properties. These glycolipid fractions were then analysed by thin-layer chromatography for Lewis activity with antibodies reactive to the type 1 precursor (Lec), H type 1 (Led), Lea and Leb epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and EI-MS/MS-TOF) and proton NMR spectroscopy. EI-MS/MS-TOF allowed for the identification of trace substances in fractions containing several other glycolipid species. Consistent with the red cell phenotype, large amounts of lactotetraosylceramide (Lec-4) were detected. Inconsistent with the red cell phenotype, small quantities of Lea-5, H-5-1 and Leb-6 glycolipids were immunochemically and structurally identified in the small intestine of this individual. By EI-MS/MS-TOF several large glycolipids with 9 and 10 sugar residues were also identified. The extensive carbohydrate chain elongation seen in this individual with a Lewis negative nonsecretor phenotype supports the concept that Lewis and Secretor blood group fucosylation may be a mechanism to control type 1 glycoconjugate chain extension. Abbreviations: FUT1, H gene; FUT2, Secretor gene, (gene bank accession no. U17894); FUT3, Lewis gene or Fuc-TIII gene, (gene bank accession no. X53578); FUT5, Fuc-TV gene; [Imm]+, immonium ion; Lea-5, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Leb-6, Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lec-4, Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Led or H-5-1, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lex-5, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; MAb, monoclonal antibody; MS, mass spectrometry; CID, collision-induced dissociation; EI, electron impact ionisation; MS/MS-TOF, tandem mass spectrometry using a time-of-flight mass spectrometer as the second mass spectrometer: m/Cz, mass-to-charge ratio; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; TLC, (high performance) thin layer chromatography. Saccharide types are abbreviated to Hex for hexose, HexNAc for N-acetylhexosamine and dHex for deoxyhexose (fucose). Ceramide is abbreviated to Cer, and ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids

Detection and Characterization of Lewis Antigens in Plasma of Lewis‐Negative Individuals Evidence of Chain Extension as a Result of Reduced Fucosyltransferase Competition

Nonacid plasma glycolipids from Lewis‐negative individuals of nonsecretor, partial‐secretor and secretor phenotypes were prepared and separated by thin‐layer chromatography and immunostained with radiolabelled Lewis antibodies. Lewis‐positive plasma and intestinal epithelial cell glycolipids from Caucasians representing the four recognized Lewis and secretor combined phenotypes were used as controls. By presenting these purified total glycolipids in a cell‐free environment to Lewis antibodies we were able to demonstrate the presence of small amounts of Lewis antigens in Lewis‐negative individuals. It is shown that lactotetraosylceramide and extended precursor glycolipids are present in all Le(a–b–) nonsecretors. Leawas detected in 1 of the 3 Le(a–b–) nonsecretor plasmas and in the intestinal sample of the same phenotype. Lactotetraosylceramide was absent but H type 1 and Lebwere both present in all group O Le(a–b–) secretors, and extended H type 1 reactive structures were also found in the partial secretor. These results clearly demonstrate that although the Lewis‐negative phenotype exists at the serological level, this phenotype is not an ‘all‐or‐nothing’ phenomenon at the chemical level. We also show that in the presence of reduced fucosyltransferase activity, increased elongation of the precursor chain occurs, which allows us to postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Lec), H type 1 (Led), Lea and Leb epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Leb blood group glycolipids (Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals.