Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Successful ABO-incompatible liver transplantation using A2 donors.

INTRODUCTION: The longer waiting time for a liver graft among patients with blood group O makes it necessary to expand the donor pool for these patients. We herein have reported our experience with ABO-incompatible liver transplantation using A(2) donors to blood group O recipients. PATIENTS AND METHODS: Between 1996 to 2005, 10 adult blood group O recipients received 10 A(2) cadaveric grafts. Mean recipient age was 52 +/- 7.7 years (mean +/- SD). The initial immunosuppression was induction with antithymocyte globulin (n = 2), interleukin-2-receptor antagonists (n = 3), or anti-CD20 antibody (rituximab, n = 1), followed by a tacrolimus-based protocol. No preoperative plasmapheresis, immunoadsorption, or splenectomies were performed. RESULTS: Patient and graft survival was 10/10 and 8/10, respectively, at 8.5 months median follow-up (range 10 days to 109 months). Two patients were retransplanted because of bacterial arteritis (n = 1) and portal vein thrombosis (n = 1). The six acute rejections, which occurred in four patients, were all reversed by steroids or increased tacrolimus dosages. The pretransplant anti-A titers against A(1) red blood cells were 1:128 (NaCl technique) and 1:8 to 1024 (IAT technique). The maximum postoperative titers were 1:64 to 4000 (NaCl) and 1:256 to 32000 (IAT). CONCLUSION: The favorable outcome of A(2) to O grafting, with a patient survival of 10/10 and graft survival of 8/10, makes it possible to consider this blood group combination also in nonurgent situations. There was no hyperacute rejection or increased rate of rejections. Anti-A/B titer changes seem to not play a significant role in the monitoring of A(2) to O liver transplantation.

Adaptation of mass spectrometry for the analysis of tumor antigens as applied to blood group glycolipids of a human gastric carcinoma.

Total neutral (nonacidic) glycolipid fractions have been isolated from a gastric adenocarcinoma and the surrounding normal gastric tissue removed at laparotomy of a Blood Group B human individual. Each glycolipid mixture was fractionated by silicic acid column chromatography into ten different partly purified glycolipid fractions. These fractions were analyzed by thin-layer chromatography and tested for Blood Group A and B activity. Blood group B activity was found in several fractions from both tumor and normal tissue. Two of the tumor glycolipid fractions reacted with some batches of commercial anti-A antisera, but other antisera tested did not react. No such Blood Group A reactivity was found in the fractions from the normal gastric tissue. The two Blood Group A-active tumor glycolipid fractions were methylated and methylated-reduced, and these two derivatives were analyzed by mass spectrometry. It was shown that these two fractions were mixtures of glycosphingolipids with five to nine sugars. The dominating glycosphingolipids were blood group Leb and B-like hexaglycosylceramides, a B-similar heptaglycosylceramide with an additional fucose, an H-like heptaglycosylceramide, and a Leb-like octaglycosylceramide. Evidence for small amounts of a Blood Group A-similar heptaglycosylceramide with an additional fucose was also found. The finding of a Blood Group A-similar glycolipid in a fraction which reacts with some anti-A antisera is the first chemical evidence for a heterolog blood group antigen in human cancer which has previously been found by histoimmunological techniques. The clinical significance of this finding is discussed in relation to diagnostic procedures and immunotherapy.

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney.

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.

Glycosphingolipids of a green monkey kidney cell line (GMK AH-1). Evidence for a novel pentaglycosylceramide based on globotetraosylceramide.

Total non-acid glycolipid fractions have been isolated from GMK AH-1 cells grown in fetal calf serum and in horse serum. For comparison, glycolipids were also prepared from green monkey (Cercopithecus aetiops) kidney and from fetal calf serum. The major glycolipids from GMK AH-1 cells grown in fetal calf serum were isolated by silicic acid column chromatography and preparative thin-layer chromatography. These fractions were characterized mainly by thin-layer chromatography, mass spectrometry and gas chromatography. The structures of the glycolipids isolated were proposed as: Glc1 leads to 1Cer, Gal1 leads to 1Cer, Gal1 leads to 4Glc1 leads to 1Cer, Gal1 leads to 4Gal1 leads to 4Glc1 leads to 1Cer, GalNAcl leads to 3Gal1 leads to 4Gal1 leads 4Glc1 leads to 1Cer. In addition, a novel pentaglycosylceramide with the probable structure Ga1 beta 1 leads to 3GalNAc beta 1 leads to Gal alpha 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer was also present. THe ceramides contained mainly dihydroxy 18:1 long-chain base in combination with non-hydroxy 16:0-24:0 fatty acids. Small amounts of trihydroxy 18:0 long-chain base and hydroxy 22:0-24:0 fatty acids were also present in the mono- and diglycosylceramide fractions. The glycolipid patterns of GMK AH-1 cells grown in fetal calf serum or horse serum were identical. The pentaglycosylceramide present in the cultured cells could not be detected with certainty in the kidney tissue. The uptake of this glycolipid from the culture medium is unlikely as it seems to be lacking in calf serum.

Glycolipids of rat small intestine. Characterization of a novel blood group H-active triglycosylceramide.

A novel blood group H-active triglycosylceramide has been isolated from rat small intestine. It was present exclusively in the epithelial cells. The structure was established by mass spectrometry, NMR spectroscopy and degradative methods to the Fucp alpha 1 leads to 2Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The lipophilic part was made up of mainly trihydroxy base (phytosphingosine) and 16 : 0--24 : 0 fatty acids.

Studies on differentiating epithelial cells of rat small intestine. Alterations in the lipophilic part of glycosphingolipids during cell migration from crypt villus tip.

Epithelial cells of rat small intestine have been separated into three intervals of different maturity correlated to cell migration from the crypt to the villus tip. The total acid and non-acid glycosphingolipids were isolated and analysed by thin-layer chromatography. The amount of glucosylceramide an N-glycoloylneuraminosyllactosylceramide was higher, while the amount of globotriaosylceramide and tetrahexosylceramide was lower in villus tip cells (more differentiated) compared to crypt cells (less differentiated). In addition to these alterations the lipophilic composition changed, as shown by a comparison by mass spectrometry of permethylated and LiAlH4-reduced, permethylated derivatives of two of the non-acid glycolipid mixtures (crypt cells and villus tip cells). The components of ceramide were mainly trihydroxy 18:0 long-chain base (phytosphingosine) and hydroxy and non-hydroxy fatty acids. The only significant change concerned the fatty acids. In the crypt cell glycolipids the most abundant fatty acid was 20:0 non-hydroxy fatty acid. In the villus tip cells there was a relative increase of hydroxy fatty acids, with the 24:0 species in dominance. This change occurred for most glycolipids, but the fatty acids of glucosylceramide were villus tip-like already in the crypt cells. The blood group A-active tetraglycosylceramide, and probably the hematoside, did not show any alteration in the lipophilic part. The results indicate that the turnover of some glycolipids (or only their lipophilic part) is more rapid than the epithelial cell turnover.

Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.

A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies.

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.

alpha-Gal epitopes in animal tissue glycoproteins and glycolipids.

alpha-Gal terminated saccharides are present on the cell surface both as glycolipids and glycoproteins in all mammals except Old World monkeys and humans. The structural diversity among identified saccharides terminated by this epitope in animal tissues is steadily increasing. The majority of these saccharides have the alpha-Gal linked to lactosamine but other core saccharides exist. The alpha-Gal terminated saccharides are recognized by the immune system as a specific antigen and antibodies directed to the alpha-Gal, which do not cross-react with the classic blood group B trisaccharide, are found in man and Old World monkeys. Similar to other complex carbohydrate cell surface antigens, the alpha-Gal epitope is heterogeneously distributed in different organs and in different cells within an organ. It is present on the vascular endothelium and it is the primary target for human naturally occurring antibodies following pig to primate/man xenotransplantation leading to hyperacute rejection of the graft. Important for the future will be to further structurally characterize this antigen system, its cellular/subcellular distribution, and to identify possible of additional glycosyltransferases, related to the already described alpha 1,3galactosyltransferase that may explain the structural diversity. Such information will be of importance in the studies of, for example, the pathogenesis of autoimmune diseases and for the production of genetically modified pigs to prevent xenograft rejection.

Serological and immunochemical characterization of anti-PP1Pk (anti-Tja) antibodies in blood group little p individuals. Blood group A type 4 recognition due to internal binding.

Serum samples from 13 blood group little p individuals were tested by radioimmunoassay for their IgG antibody subclass distribution against the P, P1 and Pk antigens. There was no uniform subclass distribution pattern, although all but one had IgG3 antibodies against all the P system antigens tested. Studies were performed adsorbing anti-Tja serum sequentially to columns with synthetic carbohydrate antigenic determinants within the P system coupled to silica beads (SynsorbsR). The effect on agglutinin and indirect antiglobulin titers was determined after adsorption to SynsorbsR with different P-system antigens (P1, Pk, P). Adsorption to all the three SynsorbsR was needed to eliminate or strongly reduce antibody titers. The effect on IgM, IgG, IgA as well as IgG subclass antibody binding to P, P1 and Pk antigens was also determined by radioimmunoassay and chromatogram binding assay. Anti-PP1Pk antibodies from a little p woman with repeated abortions were shown to bind to glycosphingolipid antigens prepared from one of the aborted placentae using a chromatogram binding assay. This binding was eliminated by serum adsorption to SynsorbsR with P1, Pk and P carbohydrates. Anti-PP1Pk antibodies were also shown to bind to extended structures in the globoseries, i.e. globopentaosylceramide, globohexaosylceramide (globo-H) and globoheptaosylceramide (globo-A). This binding is most probably due to antibodies recognizing internal sequences in the carbohydrate chain. Attempts were made to visualize the binding epitope of the antibodies by computer molecular modelling.

Characterisation of the anti-A antibody response following an ABO incompatible (A2 to O) kidney transplantation.

Anti-A,B antibodies produced in a blood group OLe(a-b-) recipient receiving a kidney graft from a blood group A2Le(a-b+) donor have been analysed for their ability to bind to different glycosphingolipid antigens. Solid-phase RIA using pure glycosphingolipid antigens and a chromatogram binding assay using total nonacid glycosphingolipid fractions from erythrocytes of different human blood group phenotypes together with pure glycolipid antigens were used as assay systems. Serum antibodies were shown to bind equally well to A (types 1, 2, 3 and 4) and B (types 1 and 2) antigenic structures but no binding to H antigens (types 1, 2 and 4) was detected. After adsorption of serum antibodies on A1 Le(a-b+) erythrocytes there was a residual anti-A antibody activity which could not be adsorbed by synthetic A-trisaccharides coupled to crystalline silica (Synsorb-A). These residual antibodies, which are not present in a pretransplant serum sample, had a specificity for the A antigen with type 1 core saccharide chain and the binding epitope obviously included both the N-acetylgalactosamine and the N-acetylglucosamine. The fucose residue was apparently not obligate for binding. The conformation of the sugar units involved in the binding epitope was determined.

Structural characterization of a blood group A heptaglycosylceramide with globo-series structure. The major glycolipid based blood group A antigen of human kidney.

A blood group A glycosphingolipid with the globo-series structure has been isolated from human kidney and structurally characterized. The structure was shown by mass spectrometry and proton NMR spectroscopy of the intact permethylated and permethylated-reduced derivatives together with degradation studies to be, GalNAc alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 Ceramide. This glycolipid reacts with both polyclonal and monoclonal anti-A blood group typing antisera and it is the major glycolipid based blood group A antigen present in the human kidney.

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors.

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.

ABO-incompatible kidney transplantation (A2 to O). Qualitative and semiquantitative studies of the humoral immune response against different blood group A antigens.

The humoral immune response against blood group A antigens with different core saccharide structures has been investigated in four blood group O recipients transplanted with kidneys from two blood group A2 donors. Radioimmunoassay and thin-layer chromatogram binding assay studies showed that different individuals responded differently to the same antigenic stimulus. Antibodies were produced in the recipient that bound to the terminal trisaccharide of the blood group A antigens. In some cases antibodies that bound to a larger antigen epitope, including the fourth and fifth sugar in the polysaccharide core chain, also occurred. Immunoglobulin class-specific, as well as subclass specific, responses were seen. The antibody response in the blood group O recipients receiving an A2 graft seem to be dependent on the antigenic expression in the transplanted kidney. In view of the recent findings of individuality of A antigen expression in kidneys within the A1 and A2 subgroups, an extended typing of A2 donors may be important. The humoral immune response in the recipient may also be dependent on earlier contacts with ABO incompatible pregnancies, vaccinations, or infections. A possible correlation between pre- and posttransplant findings was noted in one case and deserves further notice.

Heterogeneous expression of Gal alpha1-3Gal xenoantigen in pig kidney: a lectin and immunogold electron microscopic study.

BACKGROUND: The Gal alpha1-3Gal antigen (Gal alpha) is the primary target for human natural anti-pig xenoantibodies. The presence of Gal alpha has been shown in porcine endothelial cells (ECs) using light microscopy, whereas the expression of Gal alpha in other cell structures in the porcine kidney is only partially characterized. METHODS: Immunogold electron microscopy of pig kidney cryosections was performed using Griffonia simplicifolia isolectin B4 and affinity isolated human anti-Gal alpha1-3Gal antibodies. RESULTS: The most intense expression of Gal alpha was found on the apical and basolateral portions of the plasma membrane of the proximal convoluted tubule segments 1 and 2 cells, whereas segment 3 and 4 cells were negative. A strong staining was found in peritubular capillary ECs and in the inner medullary and papillary collecting duct cells. Moderate labeling of ECs and subendothelium was observed in large blood vessels, whereas glomerular ECs reacted weakly. Additionally, glomerular parietal epithelial cells, connecting tubule cells, and some cortical collecting duct cells were labeled. Among interstitial cells, a part of type-1 cells and all type-2 cells were labeled, whereas others were negative. CONCLUSIONS: By immune electron microscopy, a detailed information of the Gal alpha antigen distribution in porcine nephrons and blood vessels has been revealed, which clarifies conflicting data obtained by light microscopy. In addition, expression of the Gal alpha antigen in the renal interstitial cells was documented for the first time. These data are of importance for the understanding of xenoantibody-mediated hyperacute rejection, for interpretation of pig kidney xenograft biopsies, and for generating transgenic pigs lacking the Gal alpha epitope.

Distribution of the Galalpha1-3Gal antigen in cultured adult and fetal porcine pancreatic islet cells: an immunoelectron microscopic study.

The distribution of the Galalpha1-3Gal antigen (Galalpha) in cultured adult porcine islets (API) and fetal porcine pancreatic islet-like cell clusters (ICC) was studied using immunoelectron microscopy. API and ICC were cultured for 1 and 5 days, respectively, and immunogold labeled using human affinity isolated anti-Galalpha1-3Gal antibody, GS-IB4 lectin and antibodies against islet pancreatic hormones, vimentin, and von Willebrand factor. Differentiated endocrine cells were Gala-negative, but, in ICC, some immature endocrine cells were slightly Gala-positive. The Gala-expression in API was much weaker compared to ICC. In both API and ICC, the Gala antigen was expressed on duct epithelial cells, acinar cells, and endothelial cells. In ICC, strong Gala expression was observed on flattened cells covering their surfaces. These cells were identified as centroacinar cells originating from intra-islet ducts. In conclusion, although mature endocrine cells of cultured API and ICC lack the Gala-xenoantigen, several other cellular compounds are strongly Gala positive, which may contribute to xenorejection of these grafts.

Electron ionization-tandem mass spectrometry of glycosphingolipids. I: The identiftcation of compound-specific sequence ions in the collision-induced dissociation spectra of the immonium ions of two isomeric hexaglycosylceramides.

A permethylated-reduced hexaglycosylceramide in a complex glycolipid mixture isolated from a unique human tissue has been identified by using tandem mass spectrometry (MS/MS). The mass spectrum of this glycolipid mixture, obtained by using in-beam electron ionization, is very complex, and fragment ions derived from the hexaglycosylceramide cannot be distinguished from other ions. Tandem mass spectrometry using a four-sector mass spectrometer gave the mass spectrum of the immonium ion of the permethylated-reduced hexaglycosykeramide (m / z 1645.8), which is characteristic of its structure. Comparison of this MS/MS spectrum with those of two similarly derivatized blood group hexaglycosylceramide isomers permitted identification of the unknown glycolipid structure.

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures.

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.