Molecular cloning of a cDNA encoding a novel human leukocyte alpha-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x determinant.
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Amino Acid Sequence↗
Biomedical subjects
J B Lowe
Publications and source records attributed to J B Lowe.
General practitioner and patient response during a public education program to encourage skin examinations.
OBJECTIVE: To describe the response of general practitioners (GPs) and patients during the 1991 National Skin Cancer Awareness Week media campaign. DESIGN: Skin examinations in general practices in three representative regional towns in Queensland were monitored for five weeks around the time of the campaign and participating GPs were personally interviewed. OUTCOME MEASURES: Number and type of consultations in which the skin was examined for cancer before, during and after the campaign, and GPs' attitudes and beliefs about skin checks. RESULTS: The 46 GPs (47%) who participated, representing 60% of the practices, conducted 1805 consultations in which the skin was examined for cancer. The number of consultations in which skin examinations were conducted by each GP increased by 56% during the campaign. Skin checks were nearly always initiated by the patient (90%), and in only half of all cases was the examination given as the primary reason for the consultation. Sixty-two per cent of lesions were considered to be clinically benign. CONCLUSION: Public education programs may have an impact on the level of skin examinations requested in general practice consultations. Currently, patients are the principal initiators of such examinations during consultations--there is potential to increase the active role played by GPs in support of patient requests for the early detection of skin cancer.
Attitude to Health↗
Molecular basis for H blood group deficiency in Bombay (Oh) and para-Bombay individuals.
The penultimate step in the biosynthesis of the human ABO blood group oligosaccharide antigens is catalyzed by alpha-(1,2)-fucosyltransferase(s) (GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase, EC 2.4.1.69), whose expression is determined by the H and Secretor (SE) blood group loci (also known as FUT1 and FUT2, respectively). These enzymes construct Fuc alpha 1-->2Gal beta-linkages, known as H determinants, which are essential precursors to the A and B antigens. Erythrocytes from individuals with the rare Bombay and para-Bombay blood group phenotypes are deficient in H determinants, and thus A and B determinants, as a consequence of apparent homozygosity for null alleles at the H locus. We report a molecular analysis of a human alpha-(1,2)-fucosyltransferase gene, thought to correspond to the H blood group locus, in a Bombay pedigree and a para-Bombay pedigree. We find inactivating point mutations in the coding regions of both alleles of this gene in each H-deficient individual. These results define the molecular basis for H blood group antigen deficiency in Bombay and para-Bombay phenotypes, provide compelling evidence that this gene represents the human H blood group locus, and strongly support a hypothesis that the H and SE loci represent distinct alpha-(1,2)-fucosyltransferase genes. Candidate sequences for the human SE locus are identified by low-stringency Southern blot hybridization analyses, using a probe derived from the H alpha-(1,2)-fucosyltransferase gene.
ABO Blood-Group System↗
Molecular cloning of a cDNA encoding a novel human leukocyte alpha-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x determinant.
The sialyl Lewis x determinant (NeuAc alpha 2,3Gal beta 1, 4[Fuc alpha 1,3]GlcNAc) is an essential component of leukocyte counterreceptors for E-selectin and P-selectin. The final step in sialyl Lewis x synthesis is catalyzed by alpha-1,3-fucosyltransferases acting on sialylated glycoconjugate precursors. Cultured human leukocytic cell lines express an alpha-1,3-fucosyltransferase gene termed Fuc-TIV or ELFT but do not express the other three cloned human alpha-1,3-fucosyltransferase genes to any significant degree. The physiological role of Fuc-TIV/ELFT in sialyl Lewis x biosynthesis is uncertain, however, since it can catalyze the synthesis of this determinant in some, but not all, transfected cell lines in a manner that is dependent upon the glycosylation phenotype of the host cell. We report here the molecular cloning of a cDNA encoding a new human leukocyte alpha-1,3-fucosyltransferase, termed Fuc-TVII, capable of synthesizing the sialyl Lewis x moiety. The cDNA sequence predicts a 341-amino acid-long type II transmembrane protein typical of mammalian glycosyltransferases. When expressed in mammalian cells, the Fuc-TVII cDNA directs the synthesis of cell surface sialyl Lewis x moieties but not the Lewis x, Lewis a, sialyl Lewis a, or VIM-2 determinants. Fuc-TVII can efficiently utilize alpha-2,3-sialyllactosamine in vitro to form the sialyl Lewis x tetrasaccharide but does not utilize lactosamine to form the Lewis x moiety. Northern blot analyses show that the Fuc-TVII gene is transcribed in HL-60 cells, a human promyelocytic cell line, and in YT cells, a natural killer-like cell line. Fuc-TVII represents a leukocytic alpha-1,3-fucosyltransferase that can participate in selectin ligand synthesis via its ability to catalyze the synthesis of sialyl Lewis x determinants.
Amino Acid Sequence↗
Molecular cloning of a murine N-acetylgalactosamine transferase cDNA that determines expression of the T lymphocyte-specific CT oligosaccharide differentiation antigen.
Two monoclonal antibodies termed CT1 and CT2 define a cell surface oligosaccharide molecule expressed on restricted populations of murine lymphocytes. This oligosaccharide structure is largely associated with the extracellular domain of the CD45 family of tyrosine phosphatases, molecules required for lymphocytes to proliferate in response to antigen stimulation. Previous work has shown that the oligosaccharide structure recognized by the CT antibodies is identical, at least in part, to that of the human Sda blood group, a structure formed through enzymatic addition of N-acetylgalactosamine in beta 1,4-linkage to a sub-terminal galactose substituted with an alpha 2,3-linked N-acetylneuraminic acid residue. We have used a mammalian transient expression cloning approach to isolate a murine cDNA that determines expression of an oligosaccharide structure recognized by the CT antibodies as well as human anti-Sda serum. The nucleotide sequence predicts a 510-amino acid type II transmembrane protein characteristic of other mammalian glycosyltransferases. Enzymatic characterization of the protein expressed by this cDNA demonstrates that it encodes a beta 1,4-N-acetylgalactosamine transferase activity that can add to the low molecular weight acceptor 3'-sialyl-N-acetyllactosamine, to form the nonreducing terminal tetrasaccharide Sda blood group structure. This cDNA shares 51% nucleotide sequence identity with a cDNA encoding the human GM2/GD2 synthase, particularly throughout the regions encoding their putative catalytic domains. Southern blot analysis demonstrates that these two cDNA's represent distinct loci in the murine genome. The CT-GalNAc transferase cDNA isolated here represents a tool with which to define the role(s) of lymphocyte cell surface CT determinants, and may facilitate the isolation of the human Sda blood group locus through cross-hybridization approaches.
Amino Acid Sequence↗
Molecular basis for plasma alpha(1,3)-fucosyltransferase gene deficiency (FUT6).
While most humans express an alpha(1,3)-fucosyltransferase in plasma, 9% of individuals on the isle of Java (Indonesia) do not express this enzyme. Ninety-five percent of these plasma alpha(1,3)-fucosyltransferase-deficient individuals have Lewis negative phenotype on red cells, suggesting strong linkage disequilibrium between these two traits. To define the molecular basis for this plasma deficiency and to determine which of two candidate human alpha(1,3)-fucosyltransferase genes encode this enzyme (FUT5 and FUT6), we cloned and analyzed alleles at these two loci from an Indonesian individual deficient in plasma alpha(1,3)-fucosyltransferase activity. Single base pair changes were identified in the coding region of each gene, relative to previously published wild type alleles. These changes in turn yield three codon changes in FUT5 and three in FUT6. The codon changes in the FUT5 gene do not yield detectable diminutions in alpha(1,3)-fucosyltransferase activity when tested by expression in transfected COS-1 cells, and none of the FUT5 alleles co-segregate with plasma alpha(1,3)-fucosyltransferase deficiency in Indonesian pedigrees. By contrast, two of the codon changes in the FUT6 alleles inactivate this gene when tested by expression in transfected COS-1 cells. One of these inactivating changes is a missense mutation (Glu-247-->Lys) within the enzyme's catalytic domain. The other inactivating mutation represents a nonsense mutation (Tyr-315-->stop) that truncates the COOH terminus of the enzyme by 45 amino acids. The Glu-247-->Lys missense mutation is present in double dose in the nine plasma alpha(1,3)-fucosyltransferase-deficient individuals tested, whereas the nonsense mutation at tyrosine 315 is present in double dose in just one of these persons. These results demonstrate that the alpha(1,3)-fucosyltransferase activity in human plasma is encoded by the FUT6 gene and that the missense mutation within codon 247 of this gene is responsible for deficiency of this activity in these Indonesian families.
Alleles↗
Skin cancer prevention: a link between primary prevention and early detection?
The aim of this paper was to determine if there is any link between primary prevention and early detection for skin cancer. Results from a study of a large random sample of Gold Coast residents (N = 995) identified an association (P < 0.01) between individual primary prevention and early detection activities. People were also more likely to use both prevention methods if they had personal experience with skin cancer (P = 0.01) or if they were male (P = 0.05). Future primary prevention and early detection skin cancer programs might be most effective if they are combined.
Adolescent↗
Enhancing the early detection of melanoma within current guidelines.
Queensland has the highest reported incidence of malignant melanoma in the world, and early detection offers the possibility of reducing the burden of this cancer in the short term. We report on a survey of 995 residents of the Gold Coast, Queensland, regarding behavioural aspects of the early detection of skin cancer. Survey results suggest that although many people report some form of skin examination behaviour, by either checking their own skin, having another nonmedical person check their skin, or by going to their general practitioner for a skin check, much of this activity may be inadequate. They indicate that current early detection could be enhanced by encouraging individuals and general practitioners to extend the coverage of the examinations they already conduct.
Adolescent↗
The gene encoding myeloid alpha-3-fucosyl-transferase (FUT4) is located between D1 1S388 and D11S919 on 11q21.
The last step in the biosynthesis of Le(x) antigen, the addition of a fucose to precursor polysaccharides, can be catalyzed by different alpha-3-fucosyltransferases. We localized the gene (FUT4) encoding myeloid alpha-3-fucosyltransferase by PCR assay using panels of somatic cell and radiation hybrids which retain different rearrangements of chromosome 11. FUT4 was assigned to chromosome band 11q21 between D11S388 and D11S919.
Animals↗
Expression of alpha-(1,3)-fucosyltransferases which synthesize sialyl Le(x) and sialyl Le(a), the carbohydrate ligands for E- and P-selectins,in human malignant cell lines.
Transcripts of alpha-(1,3)-fucosyltransferases in human epithelial cancer and leukemia cell lines were analyzed by Northern blotting and reverse transcriptase mediated-polymerase chain reaction using specific probes and primers which can discriminate between the transcripts derived from the four alpha-(1,3)-fucosyltransferase genes Fuc-TIII, IV, V, and VI. Flow cytometric analysis of the sialyl Le(x) and sialyl Le(a) antigens was also performed on the same cell lines. The sialyl Le(x) antigen was expressed on 14 of 15 epithelial cancer cell lines, and the sialyl Le(a) antigen was detected on 8 of them. The message of Fuc-TIII was detected in most of the epithelial cancer cell lines (14 of 15), which correlated with the surface expression of these carbohydrate determinants. In addition, the messages of Fuc-TIV and Fuc-TVI were detected in most epithelial cancer cell lines, while the message of Fuc-TV was undetectable in most of them. On the other hand, all leukemia cell lines were positively stained for sialyl Le(x), but none of them was stained for sialyl Le(a) in flow cytometry. The messages of Fuc-TIV++ were detected in all leukemia cell lines tested. Small quantities of Fuc-TIII, V, and/or VI messages were also detected in some leukemia cell lines in reverse transcriptase mediated-polymerase chain reaction analysis. These studies indicate that alpha-(1,3)-fucosyltransferase activities in epithelial cancer and leukemia cell lines are mixtures of multiple molecular species of alpha-(1,3)-fucosyltransferases. It is natural that epithelial cancer cells contain a significant amount of Fuc-TIII mRNA and leukemia cell lines contain Fuc-TIV mRNA, since their normal counterparts, normal epithelial cells and leukocytes, respectively, are known to contain these fucosyltransferases. The unexpectedly frequent occurrence of Fuc-TIV mRNA in epithelial cancer cell lines may be related to their retro-differentiation associated with tumorigenesis. Another unexpected finding was a weak but significant expression of the alpha-(1,3)-fucosyltransferases Fuc-TIII, V, and/or VI in leukemia cell lines detected by reverse transcriptase mediated-polymerase chain reaction analysis. Since these enzymes are known to be capable of synthesizing the sialyl Le(x) determinant, this finding implies a possibility that some of them may be involved in the synthesis of sialyl Le(x) in leukemia cells.
Adenocarcinoma↗
Protective effects of sialylated oligosaccharides in immune complex-induced acute lung injury.
Using sialyl Lewisx (SLX) oligosaccharides derived from fucosyl transferase-expressing cells or generated synthetically, the ability of these compounds to protect against acute lung damage after deposition of immunoglobulin (Ig)G or IgA immune complexes has been determined. The synthetic compounds were tetra- and pentasaccharide derivates of SLX as well as the nonfucosylated forms of SLX as controls. In the IgG immune complex model of lung injury, which is E-selectin dependent, SLX preparations provided dose-dependent protective effects, as assessed by changes in lung vascular permeability and hemorrhage. Protective effects were associated with diminished tissue accumulation of neutrophils in lungs (as assessed by myeloperoxidase). Morphological assessment revealed reduced physical contact of neutrophils with the pulmonary vascular endothelium and reduced tissue accumulation of neutrophils. In the model of IgA immune complex-induced lung injury, which does not involve participation of neutrophils and is independent of the requirement for E-selectin, SLX preparations were not protective. These data suggest that, in neutrophil-mediated and E-selectin-dependent lung injury, SLX preparations provide significant, protective effects against inflammatory vascular injury. The ability to achieve antiinflammatory outcomes in vivo with appropriate oligosaccharides suggests a new approach to the blocking of acute inflammatory responses.
Acute Disease↗
Protective effects of oligosaccharides in P-selectin-dependent lung injury.
Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules, including selectins (E,P,L), which are reactive with oligosaccharides, and the family of beta 2 integrins which are reactive with endothelial intercellular adhesion molecules. These processes result in the initial rolling of leukocytes along the endothelial surfaces, followed by the firm attachment of leukocytes to the endothelium. The intravenous infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical mediated and P-selectin-dependent. Here we report that infusion of sialyl-Lewis X, a ligand for P-selectin, dramatically reduced lung injury and diminished the tissue accumulation of neutrophils, whereas irrelevant oligosaccharides had no such effects. These results suggest that sialyl-Lewis X carbohydrates may be used as a new strategy for anti-inflammatory therapy.
Animals↗
E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels.
Lysosomal membrane glycoprotein (lamp)-1 and lamp-2 are the most abundant glycoproteins within the lysosomal membrane. A small amount of lamp-1 and lamp-2 molecules, however, can be present on the cell surface. We have shown previously that highly metastatic colonic carcinoma L4 cells express more lamp-1 and lamp-2 on the cell surface than low metastatic SP cells (Saitoh, O., Wang, W.-L., Lotan, R., and Fukuda, M. (1992) J. Biol. Chem. 267, 5700-5711). Since lamp-1 and lamp-2 are the major carriers for poly-N-acetyllactosamines that are able to display sialyl-Le(x) termini, we sought to determine if an increased amount of lamp-1 on the cell surface would lead to increased expression of cell surface sialyl-Le(x) determinants and to the increased adhesion of those cells to E-selectin. Expression of increased amounts of lamp-1 on the cell surface was achieved either by overexpression of lamp-1 or by expressing a mutant lamp-1 molecule preferentially at the plasma membrane, rather than in lysosomes. Cells that express variable amounts of cell surface lamp-1 were tested for their adhesion to activated endothelial cells or E-selectin expressing Chinese hamster ovary cells. The results clearly show that the extent of adhesion to E-selectin and cell surface sialyl-Le(x) determinants is proportional to the amount of cell surface lamp-1. Moreover, it was demonstrated that such adhesion can be inhibited by soluble lamp-1 generated from Chinese hamster ovary cells expressing sialyl-Le(x) structures. These results indicate that lamp-1 can efficiently present ligands for E-selectin and at the same time can be a useful reagent for inhibition of E-selectin (and possibly P-selectin)-mediated interaction.
Animals↗
The blood group-specific human glycosyltransferases.
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ABO Blood-Group System↗
Sun-related attitudes and beliefs among Queensland school children: the role of gender and age.
A cross-sectional study of the sun-related attitudes and beliefs of 3,655 children in Grades 7 to 11 attending 55 representative Queensland schools was undertaken using a structured questionnaire administered at school. Factors derived from a series of focus groups were incorporated into the questionnaire. Knowledge about risk factors in the aetiology of skin cancer and the role of protective measures was high. Several potential barriers to the use of sun protection were identified, including the desire to be tanned, the perceived attitudes of the peer group to sun protection, and difficulties with the use of specific sun protection measures. In general, these barriers were significantly more prominent among boys and children from older grades than other students. These data will be used to design school-based sun protection interventions which are sensitive to these age and sex differences.
Adolescent↗
The dissemination of smoking cessation methods for pregnant women: achieving the year 2000 objectives.
The smoking prevalence rate among adult women and pregnant women has decreased only 0.3 to 0.5% per year since 1969. Without a nationwide dissemination of efficacious smoking cessation methods based on these trends, by the year 2000 the smoking prevalence among pregnant women will be approximately 18%. This estimate is well above the US Department of Health and Human Services Year 2000 Objective of 10%. The US dissemination of tested smoking cessation methods could help an additional 12,900 to 155,000 pregnant smokers annually and 600,000 to 1,481,000 cumulatively to quit smoking during the 1990s. Dissemination could help achieve 31 to 78% of the Year 2000 Objectives for pregnancy smoking prevalence. (With dissemination, at best a 15% smoking prevalence during pregnancy, rather than the 10% objective, is likely to be observed.) Our results confirm a well-documented need for a national campaign to disseminate smoking cessation methods.
Adult↗
Health education for pregnant smokers: its behavioral impact and cost benefit.
OBJECTIVES: A randomized trial (the Birmingham Trial II) was conducted to evaluate the behavioral impact of health education methods among 814 female smokers at four public health maternity clinics. METHODS: Four hundred patients were randomly assigned to an Experimental (E) Group, and 414 were assigned to a Control (C) Group. Self-reports and saliva cotinine tests confirmed smoking status at the first visit, at midpregnancy, and at end of pregnancy. RESULTS: The E Group exhibited a 14.3% quit rate and the C Group an 8.5% quit rate. A Historical Comparison (C) Group exhibited a 3.0% quit rate. Black E and C Group patients had higher quit rates than White E and C Group patients. A cost-benefit analysis found cost-to-benefit ratios of $1:$6.72 (low estimate) and $1:$17.18 (high estimate) and an estimated savings of $247,296 (low estimate) and $699,240 (high estimate). CONCLUSION: Health education methods are efficacious and cost beneficial for pregnant smokers in public health maternity clinics.
Adult↗
Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.
We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.
Amino Acid Sequence↗