Biomedical subjects

H Clausen

Publications and source records attributed to H Clausen.

At least 19 recordsLinked to original sources

PubMed2006

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector.

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Adenoviridae↗

PubMed2005

The risk of chronic traumatic brain injury in professional boxing: change in exposure variables over the past century.

OBJECTIVES: To determine if boxing exposure has changed over time and hence if current professional boxers are at the same risk of developing chronic traumatic brain injury (CTBI) as historical controls. DESIGN: Literature review of published studies and analysis of data of active professional boxers. SUBJECTS: Professional boxers in the United Kingdom and Australia. MAIN OUTCOME MEASURES: Boxing history and participation in sparring and professional bouts. RESULTS: Since the 1930s, the average duration of a professional boxer's career has dropped from 19 years to five years, and the mean number of career bouts has reduced from 336 to 13. This is despite no significant decline in participation rates from 1931 until 2002. CONCLUSIONS: The incidence of boxing related CTBI will diminish in the current era of professional boxing because of the reduction in exposure to repetitive head trauma and increasing medical monitoring of boxers, with preparticipation medical and neuroimaging assessments resulting in the detection of early and potentially pre-symptomatic cases of CTBI.

Australia↗

PubMed2001

Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases.

The enzymatic oxidation of uridine 5'-diphospho-alpha-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-alpha-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-epsilon-amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-alpha-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-N-acetyl-alpha-D-galactosamine (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases beta3Gal-T5, beta4Gal-T1, and beta4Gal-T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin-p-aminophenyl N-acetyl-beta-D-glucosaminide (BSA-(GlcNAc)17) and ovalbumin. The detection of the biotin tag transferred by beta3Gal-T5 onto BSA-(GlcNAc)17 with streptavidin-enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree of Gal-biotin tag transfer onto agalactosylated hybrid N-glycans present at the single glycosylation site of ovalbumin was dependent on the Gal-T used (either beta3Gal-T5, beta4Gal-T4, or beta4Gal-T1), which indicates that the acceptor specificity may direct the transfer of the Gal-biotin tag. The potential of this biotinylated UDP-Gal as a novel donor substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under-galactosylated glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.

Biotin↗

PubMed2001

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.

Amino Acid Sequence↗

PubMed2001

Distinct orders of GalNAc incorporation into a peptide with consecutive threonines.

Mucin O-glycosylation is initiated by a transfer of N-acetyl-d-galactosamine (GalNAc) to Ser and Thr residues in polypeptides with a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). In this paper, four human pp-GalNAc-Ts (pp-GalNAc-T1, T2, T3, and T4) were tested for their preferential orders of GalNAc incorporation into FITC-PTTTPITTTTK, a portion of the tandem repeat of human MUC2. The products were separated by reverse-phase HPLC and characterized by MALDI-TOF MS and peptide sequencing. pp-GalNAc-T1 showed preference for acceptor sites, but the order of the incorporation into these sites seemed to be random. In contrast, the GalNAc incorporation by pp-GalNAc-T2, T3, or T4 was not only site-specific but also according to the specific orders. Furthermore, pp-GalNAc-T2, T3, or T4 had distinct maximum numbers of GalNAc incorporations into this peptide.

Acetylgalactosamine↗

PubMed2001

Advantages of external accumulation for electron capture dissociation in Fourier transform mass spectrometry.

A combination of external accumulation (XA) with electron capture dissociation (ECD) improves the electron capture efficiency, shortens the analysis time, and allows for rapid integration of multiple scans in Fourier transform mass spectrometry. This improves the signal-to-noise ratio and increases the number of detected products, including structurally important MS3 fragments. With XA-ECD, the range of the labile species amenable to ECD is significantly extended. Examples include the first-time determination of the positions of six GalNAc groups in a 60-residue peptide, five sialic acid and six O-linked GalNAc groups in a 25-residue peptide, and the sulfate group position in a 11-residue peptide. Even weakly bound supramolecular aggregates, including nonspecific peptide complexes, can be analyzed with XA-ECD. Preliminary results are reported on high-rate XA-ECD that uses an indirectly heated dispenser cathode as an electron source. This shortens the irradiation time to > or = 1 ms and increases the acquisition rate to 3 scans/s, an improvement by a factor of 10-100.

Adrenocorticotropic Hormone↗

PubMed2001

Mass spectrometric determination of O-glycosylation sites using beta-elimination and partial acid hydrolysis.

The present study demonstrates that treating O-glycosylated peptides with methylamine vapor followed by partial acid hydrolysis is an effective means for locating O-glycosylation site(s). The reaction with methylamine transforms the glycosylated Ser and Thr residues into stable methylamine derivatives with a mass increment of +13 Da relative to nonglycosylated Ser and Thr residues. Peptide sequencing based on partial acid hydrolysis followed by mass spectrometric analysis or in favorable cases by CID-MS/MS enables the determination of the formerly O-glycosylated sites.

Acids↗

PubMed2001

A 60-kDa protein abundant in adipocyte caveolae.

To search for caveolar proteins, mice were immunised with rat adipocyte membranes. Hybridoma supernatants were screened for antibodies to proteins on the cytosolic face of caveolae by indirect immunoelectron microscopy of immunogold-labelled adipocyte plasma membrane sheets adsorbed on electron-microscope (EM) grids. One of the hybridoma supernatants (2F11) produced a specific labelling of caveolae which was much more intense than that obtained with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude membrane fractions from different rat tissues, 2F11 labelled a band corresponding to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue and in other tissues varied in parallel to caveolin- labelling. In blots of plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate of adipocytes, prior insulin stimulation of the adipocytes translocated GLUT-4 from the LM to the PM fraction, but was without effect on the distribution of the 60-kDa protein labelled by 2F11. Digestion with endoproteinase lys-C produced the same pattern of immunoreactive fragments of the protein in the vesicular PM and LM fractions, indicating similar membrane topology of the 2F11-reactive, 60-kDa protein in vesicles of PM and LM fractions.

Adipocytes↗

PubMed2001

N-acetylgalactosamine incorporation into a peptide containing consecutive threonine residues by UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases.

A limited number of glycosylation products were generated in a cell-free system from a portion of the MUC2 tandem repeat, PTTTPITTTTK, when microsome fractions of human colon carcinoma LS174T cells were used as the source of UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-T) in our previous work. The structures of all products suggested that there were only two biosynthetic pathways in the GalNAc incorporation into this peptide. In the present report, the putative biosynthetic intermediates, PTTT*PITTTTK (asterisk designates a GalNAc residue), PT*TTPITTTTK, PTT*T*PITT*T*TK, and PT*TTPIT*T*T*TK, of these two hypothetical pathways were used as acceptors to prove that these two pathways do exist. The incubation products of these glycopeptides, microsome fractions of LS174T cells, and UDP-GalNAc were fractionated by reverse-phase HPLC and their structures were determined using MALDI-TOF MS and peptide sequencing. The products from PTTT*PITTTTK were PTTT*PITTT*TK, PTTT*PITT*T*TK, PTT*T*PI-TT*T*TK, PTT*T*PIT*T*T*TK, PT*T*T*PIT*T*T*TK, and PT*T*T*PIT*T*T*T*K. The products from PTT*-T*PITT*T*TK exactly corresponded to the products with five to seven GalNAc residues from PTTT*PITTTTK. The products from PT*TTPITTTTK were PT*TTPITT*TTK, PT*TTPIT*T*TTK, and PT*TTPIT*T*T*TK. PT*TTP-IT*T*T*TK was not converted further under the applied condition. All the products detected and analyzed were the same as those obtained when the unsubstituted peptide and microsome fractions of LS174T cells were incubated. Immunocytochemical analysis indicated that LS174T cells contain at least four pp-GalNAc-Ts (-T1, -T2, -T3, and -T4), suggesting that control of the order and the maximum number of GalNAc incorporation into this peptide is regulated through the coordinated actions of these and possibly other pp-GalNAc-Ts.

Acetylgalactosamine↗

PubMed2001

Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides.

We present evidence that site-specific O-glycosylation by recombinant polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 is controlled by the primary sequence context, as well as by the position and structure of previously introduced O-glycans. Synthetic mucin-type (glyco)peptides corresponding to sections of the tandem repeat regions of MUC1, MUC2, and MUC4 were used as substrates for recombinant polypeptide N-acetylgalactosaminyltransferases, rGalNAc-T2 and -T4. By concerted and sequential action the two transferases are able to fully glycosylate MUC1 but only partially MUC2 and MUC4 tandem repeat peptides. GalNAc residues on MUC1 acceptor peptides trigger activity of rGalNAc-T4 directed to Ser in VTSA and Thr in PDTR and of rGalNAc-T2 to Ser/Thr within the GSTA motif of variant MUC1 peptides. However, elongation of GalNAc by beta3-galactosylation inhibits rGalNAc-T4 activity completely and rGalNAc-T2 activity with respect to the acceptor site GSTA. These findings are in accord with the inhibition of rGalNAc-T2 and -T4 by fully GalNAc-substituted MUC1 repeat peptide and support a glycosylation-dependent activity induction or enhancement of both enzymes.

Amino Acid Sequence↗

PubMed2000

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1.

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.

Breast Neoplasms↗

PubMed2000

The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities.

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino acid substitution in the lectin domain of a secreted form of GalNAc-T4 selectively blocked GalNAc-glycopeptide activity, while the general activity to peptides exerted by this enzyme was unaffected. Furthermore, the GalNAc-glycopeptide activity of wild-type secreted GalNAc-T4 was selectively inhibited by free GalNAc, while the activity with peptides was unaffected.

Amino Acid Sequence↗

PubMed2000

Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.

Ligands that are capable of activating Notch family receptors are broadly expressed in animal development, but their activity is tightly regulated to allow formation of tissue boundaries. Members of the fringe gene family have been implicated in limiting Notch activation during boundary formation, but the mechanism of Fringe function has not been determined. Here we present evidence that Fringe acts in the Golgi as a glycosyltransferase enzyme that modifies the epidermal growth factor (EGF) modules of Notch and alters the ability of Notch to bind its ligand Delta. Fringe catalyses the addition of N-acetylglucosamine to fucose, which is consistent with a role in the elongation of O-linked fucose O-glycosylation that is associated with EGF repeats. We suggest that cell-type-specific modification of glycosylation may provide a general mechanism to regulate ligand-receptor interactions in vivo.

Alkaline Phosphatase↗

PubMed2000

Cloning and expression of the histo-blood group Pk UDP-galactose: Ga1beta-4G1cbeta1-cer alpha1, 4-galactosyltransferase. Molecular genetic basis of the p phenotype.

The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P(1), P, and P(k) glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Galbeta1-R 4-alpha-galactosyltransferases catalyze the syntheses of the structurally related P(1) and P(k) antigens. The P(1) polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an alpha4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated alpha4Gal-T1. Expression of full coding constructs of alpha4Gal-T1 in insect cells revealed it encoded P(k) but not P(1) synthase activity. Northern analysis showed expression of the transcript correlating with P(k) synthase activity and antigen expression in human B cell lines. Transfection of P(k)-negative Namalwa cells with alpha4Gal-T1 resulted in strong P(k) expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that alpha4Gal-T1 represented the P(k) gene. Sequence analysis of the coding region of alpha4Gal-T1 in P(1)+/- individuals did not reveal polymorphisms correlating with P(1)P(2) typing.

Amino Acid Sequence↗

PubMed2000

Control of O-glycan branch formation. Molecular cloning and characterization of a novel thymus-associated core 2 beta1, 6-n-acetylglucosaminyltransferase.

Core 2 O-glycan branching catalyzed by UDP-N-acetyl-alpha-D-glucosamine: acceptor beta1, 6-N-acetylglucosaminyltransferases (beta6GlcNAc-Ts) is an important step in mucin-type biosynthesis. Core 2 complex-type O-glycans are involved in selectin-mediated adhesion events, and O-glycan branching appears to be highly regulated. Two homologous beta6GlcNAc-Ts functioning in O-glycan branching have previously been characterized, and here we report a third homologous beta6GlcNAc-T designated C2GnT3. C2GnT3 was identified by BLAST analysis of human genome survey sequences. The catalytic activity of C2GnT3 was evaluated by in vitro analysis of a secreted form of the protein expressed in insect cells. The results revealed exclusive core 2 beta6GlcNAc-T activity. The product formed with core 1-para-nitrophenyl was confirmed by (1)H NMR to be core 2-para-nitrophenyl. In vivo analysis of the function of C2GnT3 by coexpression of leukosialin (CD43) and a full coding construct of C2GnT3 in Chinese hamster ovary cells confirmed the core 2 activity and failed to reveal I activity. The C2GnT3 gene was located to 5q12, and the coding region was contained in a single exon. Northern analysis revealed selectively high levels of a 5.5-kilobase C2GnT3 transcript in thymus with only low levels in other organs. The unique expression pattern of C2GnT3 suggests that this enzyme serves a specific function different from other members of the beta6GlcNAc-T gene family.

Amino Acid Sequence↗

PubMed2000

IDDM7 links to insulin-dependent diabetes mellitus in Danish multiplex families but linkage is not explained by novel polymorphisms in the candidate gene GALNT3. The Danish Study Group of Diabetes in Childhood and The Danish IDDM Epidemiology and Genetics Group.

The insulin-dependent diabetes mellitus (IDDM) susceptibility locus IDDM7 on 2q31 links to IDDM in some but not other populations. Linkage of D2S152, the marker for IDDM7, has hitherto not been demonstrated in Danish patients. GALNT3 that encodes the UDP-GalNAc: polypeptide N-acetyl-galactosaminyltransferase-T3 (GalNAc-T3), was recently identified and mapped to a region 5-25 cM from D2S152. The GalNAc transferases may play a role in immune mediated diseases by glycosylating autoantigens. Hence, the aims of the present study were to investigate by means of extended transmission disequilibrium testing (ETDT) and transmission disequilibrium testing (TDT) of the marker for IDDM7, D2S152, the marker for GALNT3, D2S2363, and novel polymorphisms identified through mutation screening of the entire GALNT3 for linkage with IDDM in 241 Danish IDDM multiplex families. ETDT analysis demonstrated linkage between IDDM and D2S152 (P(ETDT)=0.034). A prevalent T-->A polymorphism, T284A, was found in the GALNT3 3'UTR. Analysis of the D2S2363 and the T284A GALNT3 transmission patterns did not show linkage to IDDM in Danish patients (P(ETDT)=0.15 and P(TDT)=0.76, respectively). In conclusion, IDDM7 (D2S152) links to IDDM in Danish patients, but D2S2363 and the identified T284A polymorphism in the GALNT3 3'UTR did not. Hence, it is unlikely that the GALNT3 is an IDDM susceptibility gene.

Adult↗

PubMed2000

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties.

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.

Amino Acid Sequence↗

PubMed2000

Characterization of the histo-blood group O(2) gene and its protein product.

BACKGROUND AND OBJECTIVES: This study aimed to show the full sequence and function of the O(2) allele, and investigate whether it accounts for the incompatible expression of A antigens in gastric carcinomas of blood group O persons. MATERIALS AND METHODS: By PCR, we determined the ABO genotype of group O subjects (76 gastric carcinoma patients and 165 blood donors). Two expression constructs, encoding either the putative soluble or full-length O(2) protein, were used to transfect Sf9 cells. The expression and the activity of the O(2) protein were analysed by immunohistochemistry and enzymatic assays, respectively. RESULTS: No significant difference was detectable between the O(2) allele frequency in gastric carcinoma patients (3.9%) and blood donors (4.2%). Sequencing analysis of the O(2) allele revealed an intact reading frame identical to that of A transferase except for four nucleotide substitutions. O(2)-transfected Sf9 cells and gastric carcinomas genotyped as O(1)O(2) both expressed a protein recognized by anti-A/B transferase monoclonal antibodies. In enzymatic assays, the O(2) protein failed to show measurable A transferase activity. CONCLUSION: The O(2) allele has an intact reading frame encoding a protein immunologically related to A/B transferases and enzymatically inactive. Further, our data gave no indication that the O(2) allele is related to the phenomenon of incompatible A antigen expression in gastric cancer.

ABO Blood-Group System↗