Phenotyping of Lewis and secretor HBGA from saliva and detection of new FUT2 gene SNPs from young children from the Amazon presenting acute gastroenteritis and respiratory infection.

The Histo-blood group antigens (HBGA) are host genetic factors associated with susceptibility to rotavirus (RV) and human norovirus (HuNoV), the major etiological agents of viral acute gastroenteritis (AGE) worldwide. The FUT2 gene expressing the alpha-1, 2-L- fucosyltransferase enzyme is important for gut HBGA expression, and also provides a composition of the phenotypic profile achieved through mutations occurring in populations with different evolutionary histories; as such, it can be considered a genetic population marker. In this study, Lewis and secretor HBGA phenotyping was performed using 352 saliva samples collected from children between three months and five years old born in the Amazon (Brazil, Venezuela and English Guyana) presenting AGE or acute respiratory infection (ARI), the latter considered as control samples. The total of children phenotyped as secretors was 323, corresponding to 91.80%. From these, 207 (58.80%) had a Le (a + b+) profile. The HBGA profiles were equally found in children with AGE as well as with ARI. The rs1047781 of the FUT2 gene was not detected in DNA from saliva cells with a Le (a+b+) profile. However, mutations not yet described in the FUT2 gene were observed: missense 325A>T, 501C>T, 585C>T, 855A>T and missense substitutions 327C>T [S (Ser) > C (Cys)], 446 T>C [L(Leu) > P(Pro)], 723C>A [N(Asn) > K(Lys)], 724A>T [I(Ile) > F(Phe)], 736C>A [H(His) > N(Asn)]. The SNP distribution in the FUT2 gene of the analyzed samples was very similar to that described in Asian populations, including indigenous tribes.

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(ß3)GlcNAc, Gal(ß4)GlcNAc and Gal(ß3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and ß4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(ß4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.

Establishment of a novel lectin-antibody ELISA system to determine core-fucosylated haptoglobin.

BACKGROUND: Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker that increases in various pathological conditions. We previously established a Fuc-Hpt lectin-antibody assay using Aleuria aurantia lectin (AAL), and applied this to diagnose several diseases, including various cancers. AAL recognizes both α1-3/1-4 and α1-6 fucosylation on N/O-linked glycans. These fucosylation types differ in biological function, and in regulation by different fucosyltransferases. Recently, we identified a novel lectin, Pholiota squarrosa lectin (PhoSL), which specifically recognizes α1-6 fucosylation (core-fucosylation). METHODS: We developed a lectin-antibody ELISA kit using PhoSL to determine core-Fuc-Hpt levels in sera from colorectal or pancreatic cancer patients. RESULTS: Serum levels of AAL-reactive Hpt are higher in pancreatic cancer patients, whereas those of PhoSL-reactive Hpt are higher in colorectal cancer patients. Mass spectrometry analyses of Hpt fucosylation levels were consistent with lectin-antibody ELISA results. Hpt-transfected colorectal cancer cell lines produced significant amounts of core-Fuc-Hpt, suggesting that colorectal cancer tissues produce core-Fuc-Hpt. CONCLUSIONS: These differences in Fuc-Hpt patterns might depend on cancer cells and the surrounding cells, which produce Hpt.

Fucosyltransferase IV (FUT4) as an effective biomarker for the diagnosis of breast cancer.

Specific enzymes are involved in altered glycosylation of cancer. Fucosyltransferase IV (FUT4) is associated with the proliferation and metastasis of breast cancer. The application of FUT4 assay in the serum has not been reported yet. Here, the expression level of FUT4 in the breast cancer patient's tissues (n=60) was analyzed by immunohistochemistry (IHC) and the secreted FUT4 in blood serum samples (n=225) was detected by enzyme-linked immunosorbent assay (ELISA). Using low metastatic MCF-7 and high metastatic MDA-MB-231 breast cancer cell lines, FUT4 expression was also detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunofluorescent staining. The conventional cancer biomarkers cancer antigen (CA15.3) and carcinoembryonic antigen (CEA) was analyzed by Elecsys-electrochemical immune assay (ECLIA) to compare specificity and sensitivity with that of FUT4. We have observed a significant high expression of FUT4 in breast cancer tissues and serums as compared to the normal tissues (P<0.01) and control serums (P<0.05). FUT4 expression was increased in MDA-MB-231 cells vs. that in MCF-7 cells. Furthermore, the results of receiver operating characteristic (ROC) analysis was shown, area under curve of FUT4 (AUC=0.784) was higher than that of CA15.3 (AUC=0.468) and CEA (AUC=0.563). The relation analysis is indicated FUT4 is significantly correlated with CA15.3 (r=0.234, P<0.05) and there is no significant correlation with CEA. In conclusion, this study suggests that FUT4 can serve as novel biomarker in the diagnosis and prognosis of breast cancer.

Association between circulating osteoblast progenitor cells and aortic calcifications in women with postmenopausal osteoporosis.

BACKGROUND AND AIMS: Ectopic artery calcification has been documented in women with postmenopausal osteoporosis, in whom an imbalance in the number of circulating osteoprogenitor cells (OPCs) has been identified. Circulating OPCs form calcified nodules in vitro; however, it remains unknown whether an association exists between the number of circulating OPCs and aortic calcifications. We investigated the relationship between OPCs and aortic calcifications in women with postmenopausal osteoporosis. METHODS AND RESULTS: The number of circulating OPCs was quantified by FACS analysis in 50 osteoporotic postmenopausal women. OPCs were defined as CD15-/alkaline-phosphatase(AP)+ cells coexpressing or not CD34. Participants underwent measurement of markers of bone metabolism, bone mineral density and abdominal aortic calcium (AAC) by 64-slice computed tomography. Patients with AAC were older, had lower 25(OH)vitamin D levels and higher circulating CD15-/AP+/CD34- cells than those without AAC. Significant correlates of AAC included age (rho = 0.38 p = 0.006), calcium (rho = 0.35 p = 0.01), 25(OH)vitamin D (rho = -0.31, p = 0.03) and the number of CD15-/AP+/CD34- cells (rho = 0.55 p < 0.001). In regression analyses, the log-transformed number of CD15-/AP+/CD34- cells was associated with the presence (OR = 6.45, 95% CI 1.03-40.1, p = 0.04) and severity (ß = 0.43, p < 0.001) of AAC, independent of age, 25(OH)vitamin D, calcium and other potential confounders. Patients with low 25(OH)vitamin D and high CD15-/AP+/CD34- cells had higher median AAC than other patients (1927/µL, 862-2714/µL vs 147/µL, 0-1665/µL, p = 0.003). CONCLUSION: In women with postmenopausal osteoporosis, the number of circulating CD15-/AP+/CD34- cells is significantly associated with increased aortic calcifications, that appear to be correlated also with reduced 25(OH)vitamin D levels.

Distribution and impact of erythrocyte Lewis-system antigens on patients with ischemic heart diseases in the west of Georgia.

The aim of the research is to reveal the cases of Lewis System Antigens Phenotype in West Georgia and setting the connection between antigens expressivness and IHD. Therefore, we have phenotypically tested 393 people (236 healthy donors; average age 42±7,5 and 157 patients ill with ischemic heart diseases; average age 62,5±7,5). In accordance with the findings, the number of Lewis -antigens among healthy population is 46,6% (110 ±4,8; p<0.05) with Lea-b+ phenotype; 30,9% - with Lea-b- phenotype(73±2,9; p<0.03); 19% - with Lea+b- phenotype-(47±1,7; p<0.03) Only 2,6% cases of phenotype Lea+b+ (6±0,2; p<0.02),were revealed among healthy population. As for the patients with ischemic heart diseases we got the following results: 41% cases of Lea-b- phenotype (65±3,9; p<0.05); 32,8% - Lea-b+ (51±3,2; p<0.03); 21,1% - Lea+b- - phenotype 33±2,9; p<0.03) and 5,6% - Lea+b+ phenotype (8±1,2; p<0.02). On the whole, in the West Georgia the most frequent phenotype is Lea-b+ among healthy population and Lea-b- phenotype among people with ischemic heart diseases. Research was carried out in a control group according to Lewis antigen phenotype. People were separated in two groups; I group -healthy people with Lea-b- phenotype and II group - healthy people with Lea-b+ and Lea+b- phenotypes. On the basis of the research we concluded that people in the first group (with Lea-b- phenotype) had a high BMI, arterial hypertension and lower indexes of high density lipoprotein and triglyceride than the people in the second group(with Lea-b+ and Lea+b- phenotypes. These kinds of changes (characterised to the people with Lea-b- phenotype) are associated with a high risk of ischemic diseases and atherosclerosis. To sum up, people with Lea-b- phenotype have a high risk of ischemic heart disease. In accordance with the findings, Lewis phenotype research can be carried out to detect HID and other diseases as well (hypertension, ischemic insult and insulin-dependent diabetes mellitus).

α1,6-Fucosyltransferase (Fut8) is implicated in vulnerability to elastase-induced emphysema in mice and a possible non-invasive predictive marker for disease progression and exacerbations in chronic obstructive pulmonary disease (COPD).

Fut8 (α1,6-Fucosyltransferase) heterozygous knock-out (Fut8(+/-)) mice had an increased influx of inflammatory cells into the lungs, and this was associated with an up-regulation of matrix metalloproteinases, MMP-2 and MMP-9, after treatment with porcine pancreatic elastase (PPE), exhibiting an emphysema-prone phenotype as compared with wild type mice (Fut8(+/+)). The present data as well as our previous data on cigarette-smoke-induced emphysema [8] led us to hypothesize that reduced Fut8 levels leads to COPD with increased inflammatory response in humans and is associated with disease progression. To test this hypothesis, symptomatic current or ex-smokers with stable COPD or at risk outpatients were recruited. We investigated the association between serum Fut8 activity and disease severity, including the extent of emphysema (percentage of low-attenuation area; LAA%), airflow limitation, and the annual rate of decline in forced expiratory volume in 1 s (FEV(1)). Association with the exacerbation of COPD was also evaluated over a 3-year period. Serum Fut8 and MMP-9 activity were measured. Fut8 activity significantly increased with age among the at risk patients. In the case of COPD patients, however, the association was not clearly observed. A faster annual decline of FEV(1) was significantly associated with lower Fut8 activity. Patients with lower Fut8 activity experienced exacerbations more frequently. These data suggest that reduced Fut8 activity is associated with the progression of COPD and serum Fut8 activity is a non-invasive predictive biomarker candidate for progression and exacerbation of COPD.

Investigation of unexpected serum CA19-9 elevation in Lewis-negative cancer patients.

BACKGROUND: Cancer patients with a Lewis (a-b-) phenotype have no carbohydrate antigen 19-9 (CA19-9) in their serum. However, we found a small but distinct elevation in the serum CA19-9 level in three cancer patients with the Lewis-negative phenotype. Here, we investigated the reason of such phenomena. METHODS: Six cancer patients with a Lewis-negative phenotype were selected by very low CA19-9 concentrations: three showed a small elevation (Group A) and the other three showed no elevation (Group B) in the serum CA19-9. We investigated the difference by analyzing the Lewis/Secretor genotypes. RESULTS: All of the six patients with a Le (a-b-) phenotype were genuine Le-negative genotypes: four individuals were homozygous for le1 (le(59,508)), one patient was compound heterozygous for le1 (le(59,508)) and le2 (le(59,1067)) and one patient was compound heterozygous for le1 and le(202,314). As for the Secretor gene, the three patients in Group B were homozygous for Se2 (one patient) or compound heterozygous for Se2 and sej (two patients), while the patients in Group A were all homozygous for sej genotypes. CONCLUSIONS: Even genuinely Le-negative patients, who genetically lack the Le enzyme and theoretically never produce CA19-9, occasionally show a slight increase in serum CA19-9 level when they are homozygous for Se-negative genotypes and suffer from advanced cancer with overproduction of glycans as precursors of CA19-9. Although such cases are not frequent, we should be acquainted with the correlation between serum CA19-9 values and genotypes of Lewis and Secretor genes.

alpha1-acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis.

BACKGROUND: Serum alpha1-acid glycoprotein (AGP), an acute-phase protein secreted by the liver, carries alpha(1,3)-fucosylated structures on its 5 highly branched, N-linked sugar chains. METHODS: Serum AGP levels in patients with various types of malignancies (n=214 patients) were measured using an enzyme-linked immunosorbent assay with anti-AGP antibody. To investigate glycoforms that differed in their degree of branching and extent of fucosylation, serum AGP samples were analyzed by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A, and Aleuria aurantia lectin (AAL), and anti-AGP antibody. RESULTS: A significant difference (P <0.001) in serum AGP levels was observed in preoperative patients compared with levels in the healthy control group, but the levels in individual patients did not reflect their clinical status. Conversely, it was found not only that the patterns of AGP glycoforms differed widely in the patient group compared with the healthy control group, but they also changed depending on each patient's clinical status. Furthermore, AGP glycoforms seemed to be appropriate markers of disease progression and prognosis according to follow-up studies of 45 patients during prolonged preoperative and postoperative periods. CONCLUSIONS: Patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated triantennary and tetraantennary sugar chains for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms without such changes were expected to have a good prognosis.

Chemical modifications of alpha1,6-fucosyltransferase define amino acid residues of catalytic importance.

alpha1,6-Fucosyltransferase (alpha6FucT) of human platelets was subjected to the action of phenylglyoxal (PLG), pyridoxal-5'-phosphate/NaBH(4) (PLP), and diethyl pyrocarbonate (DEPC) the reagents that selectively modify the structure of amino acids arginine, lysine and histidine, respectively, as well as to N-ethylmaleimide (NEM), mersalyl, p-chloromercuribenzoate (pCMB), iodoacetate, iodoacetamide, and methyl iodide that react with sulfhydryl group of cysteine. In addition, we treated the enzyme with beta-mercaptoethanol, a reagent that disrupts disulfide bonds. All reagents except NEM significantly inactivated alpha6FucT. Protection against the action of PLG, PLP and sulfhydryl modifying reagents was offered by GDP-fucose, GDP, and the acceptor substrate, a transferrin-derived biantennary glycopeptide with terminal GlcNAc residues. Neither donor nor acceptor substrate offered, however, any protection against inactivation by DEPC or beta-mercaptoethanol. We conclude that arginine, cysteine and probably lysine residues are present in, or closely by, the donor and acceptor substrate binding domains of the enzyme, whereas histidine may be a part of its catalytic domain. However, the primary structure of alpha6FucT does not show cysteine residues in proximity to the postulated GDP-fucose-binding site and acceptor substrate binding site of the enzyme that contains two neighboring arginine residues and one lysine residue (Glycobiol. 10 (2000) 503). To rationalize our results we postulate that platelet alpha6FucT is folded through disulfide bonds that bring together donor/acceptor-binding- and cysteine- and lysine-rich, presumably acceptor substrate binding sites, thus creating a catalytic center of the enzyme.

Molecular analysis of plasma alpha 1,3-fucosyltransferase deficiency and development of the methods for its genotyping.

Four patients with mental illness were found to be deficient in plasma alpha1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125-130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu-->247 to Lys) and C945 to A (Tyr-->315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack alpha1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of alpha1,3-fucosylated molecules on alpha(1)-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma alpha1,3-fucosyltransferase deficiency.

Plasma fucosyltransferase activity in patients with hepatocellular carcinoma, with special reference to correlation with fucosylated species of alpha-fetoprotein.

BACKGROUND/AIMS: Our previous results showed that the percentage of fucosylated species of alpha-fetoprotein (AFP) in total AFP, fucosylation index, was a very useful diagnostic tool to distinguish AFP due to hepatocellular carcinoma from AFP due to non-neoplastic liver diseases. On the other hand, alpha1-6 fucosyl-transferase (alphaFT) catalyzes the addition of fucose from GDP-fucose through an alpha1-6 linkage to the reducing end of N-acetylglucosamine residue of N-linked oligosaccharides of glycoproteins. However, the biological and clinical significance of alphaFT in patients with hepatocellular carcinoma is not fully understood. In the present study, we measured alphaFT activity to elucidate the enzymatic background of fucosylated species of AFP in hepatocellular carcinoma. METHODS: Plasma samples from 84 cases of hepatocellular carcinoma, 40 of liver cirrhosis, 40 of chronic hepatitis and 30 of normal controls, and 26 paired samples of hepatocellular carcinoma and surrounding noncancerous tissues were enrolled in the present study. AlphaFT activity was measured by high performance liquid chromatography with a synthesized fluorescence-labeled glycopeptide with an asialoagalactobiantennary sugar chain as a substrate in the presence of GDP-fucose. RESULTS: Plasma alphaFT activities (mean+/-SD, pmol/ml/h) in patients with hepatocellular carcinoma, liver cirrhosis, chronic hepatitis and normal controls were 435+/-271, 490+/-290, 590+/-209 and 380+/-133, respectively. AlphaFT levels in hepatocellular carcinoma and chronic liver diseases were increased compared with that in normal controls. A statistically significant positive correlation was observed between plasma alphaFT activity and fucosylation index of AFP (r=0.34, p= 0.0032) in 60 patients with hepatocellular carcinoma, in which increments of serum AFP were observed. When the tentative cutoff value of fucosylation index was set at 18%, which corresponded to the cutoff value to discriminate between hepatocellular carcinoma and non-neoplastic liver diseases in our previous study, the plasma alphaFT activity in hepatocellular carcinoma patients whose fucosylation index was more than 18% (n=32, 523+/-324 pmol/ml/h) was higher than that in hepatocellular carcinoma patients whose fucosylation index was equal to or less than 18% (n=28, 383+/-229) (p=0.055). An increment of the plasma levels of alphaFT occurred in accordance with an advancement of hepatocellular carcinoma stages. Tissue aFT activity in hepatocellular carcinoma (175+/-178 pmol/mg/h) was higher than those in surrounding noncancerous liver (144+/-134) and in normal liver (79+/-19). The mean alphaFT activities in well-, moderately- and poorly-differentiated hepatocellular carcinoma were 38+/-0.7, 177+/-182 and 219+/-189, respectively, and they tended to increase with dedifferentiation in hepatocellular carcinoma tissues. CONCLUSIONS: The present study indicates that alphaFT is responsible for the formation of the fucosylated species of AFP in hepatocellular carcinoma and suggests that the measurement of alphaFT provides a possible aid in the evaluation of the degree of advancement in patients with hepatocellular carcinoma.

Identification of two functionally deficient plasma alpha 3-fucosyltransferase (FUT6) alleles.

One Indonesian individual without detectable plasma alpha3-fucosyltransferase activity was identified with three point mutations, 730C>G (L244V), 907C>G (R303G), and 370C>T (P124S), in the coding region of one FUT6 allele. Another individual, expressing weak plasma alpha3-fucosyltransferase activity, had the 907C>G together with the 370C>T mutation, but did not have the 730C>G mutation. PCR-RFLP analyses of complete families confirmed the segregation of these alleles and illustrated the existence and inheritance of the [370C>T; 907C>G] mutated allele in three additional families. Altogether, this allele was found heterozygously in nine Indonesian and two Swedish individuals, all with detectable plasma alpha3-fucosyltransferase activities. The FUT6 allele with the three mutations (370C>T; 730C>G; 907C>G) was identified heterozygously in only two Indonesian individuals, both having the inactivating 739G>A mutation in the other allele and both lacking plasma alpha3-fucosyltransferase activity. Enzyme studies made on transiently transfected COS-7 cells demonstrated that the combination of the 370C>T, 730C>G and 907C>G mutations decreased the V(max) by more than 80%, but caused no obvious change of the apparent K(m) values for GDP-fucose and Gal-N-acetyllactosamine. In comparison, chimeric constructs with the isolated 730C>G or 907C>G mutations decreased the V(max) values by about two thirds and one third, respectively.

Tumor cells as the origin of elevated serum alpha1,3fucosyltransferase in association with malignancy.

We have previously reported that the elevated activities of serum alpha 1,3fucosyltransferase reverted to normal levels after curative removal of the tumors. To determine the origin of elevated serum alpha 1,3fucosyltransferase, blood samples were obtained from both the drainage vein and the artery in patients with different stages of colorectal cancer at surgery. The enzyme levels in all samples from the drainage vein were found to be higher than the levels in the artery that fed the tumor. Hence, the origin of elevated alpha1,3fucosyltransferase in serum was thought to be the tumor rather than the liver that is the normal source of serum alpha1,3fucosyltransferase. When serum samples not only from colorectal cancer patients but also from patients with gastric, liver, lung, pancreas, bladder and esophagus cancer were treated with anti-FUTVI antibody, the measured activities of alpha1,3fucosyltransferase were markedly reduced. Further, secretion of alpha1,3fucosyltransferase from human colorectal carcinoma cells was also detected in the culture medium by Western immuno-blot analysis with anti-FUTVI antibody.

On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.

The SWISS-PROT protein sequence data bank contains at present nearly 75,000 entries, almost two thirds of which include the potential N-glycosylation consensus sequence, or sequon, NXS/T (where X can be any amino acid but proline) and thus may be glycoproteins. The number of proteins filed as glycoproteins is however considerably smaller, 7942, of which 749 have been characterized with respect to the total number of their carbohydrate units and sites of attachment of the latter to the protein, as well as the nature of the carbohydrate-peptide linking group. Of these well characterized glycoproteins, about 90% carry either N-linked carbohydrate units alone or both N- and O-linked ones, attached at 1297 N-glycosylation sites (1.9 per glycoprotein molecule) and the rest are O-glycosylated only. Since the total number of sequons in the well characterized glycoproteins is 1968, their rate of occupancy is 2/3. Assuming that the same number of N-linked units and rate of sequon occupancy occur in all sequon containing proteins and that the proportion of solely O-glycosylated proteins (ca. 10%) will also be the same as among the well characterized ones, we conclude that the majority of sequon containing proteins will be found to be glycosylated and that more than half of all proteins are glycoproteins.

The alpha1-6-fucosyltransferase gene and its biological significance.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human alpha1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as alpha1-2FucT, alpha1-3FucT and alpha1-4FucT was found except for a region consisting of nine amino acids. The alpha1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The alpha1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. alpha1-6FucT is widely expressed in various rat tissues and the expression of alpha1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, alpha1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum alpha1-6-fucosylated alpha-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which alpha1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of alpha1-6FucT alone. Interestingly, when the alpha1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on alpha1-6FucT and summarize its properties, gene expression and biological significance.

Plasma alpha1,3-fucosyltransferase deficiency in schizophrenia.

Levels of plasma alpha1,3-fucosyltransferase (alpha1,3FT) were assayed in 44 patients with schizophrenia and in 50 healthy controls. Significantly reduced enzyme activities were observed in patients (p < 0.05) and 4 unrelated patients were found, for the first time in Japan, to be deficient in the enzyme activity. Two point mutations in the coding region of the FUT6 gene encoding plasma alpha1,3FT that were responsible for the inactivation of the enzyme activity were detected in those patients. Genotyping of the Le gene (FUT3) in these patients demonstrated that 2 of them were also FUT3 deficient and were grouped as Lewis- individuals whereas the rest were Lewis+.

A novel method for determination of alpha1,6fucosyltransferase activity using a reducing oligosaccharide from egg yolk as a specific acceptor.

A new method for determination of alpha1,6fucosyltransferase activity has been described. Recently, the disialyl-biantennary undecasaccharide was prepared in high yield from egg yolk [(1996), Carbohydr Lett 2: 137-42]. By treatment of this oligosaccharide with neuraminidase and beta-galactosidase, we readily obtained an asialo-agalacto-biantennary heptasaccharide (GlcNAcbeta 1,2Manalpha1,6[GlcNAcbeta1,2Manalpha1,3]Manbeta1 ,4GlcNAcbeta1,4GlcNAc). Using this asialo-agalacto-oligosaccharide as an acceptor, fucosyltransferases from human plasma and extracts of various human hepatoma cell lines were assayed in the presence of GDP-[3H]fucose. The reaction mixture was applied to a column of GlcNAc-binding, Psathyrella velutina lectin coupled gel. All the fucosylated acceptor were bound to the column which was eluted with 50 mM GlcNAc. Structural analyses revealed that only the innermost GlcNAc residue of the acceptor was fucosylated through an alpha1,6-linkage, and the oligosaccharide prepared could be used as a specific acceptor for alpha1,6fucosyltransferase. The present method was used to screen plasma alpha1,6fucosyltransferase in several patient groups, and significantly elevated activities were found in samples from patients with liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines.

P-selectin (CD62P) is a member of the selectin family of adhesion molecules involved in the regulation of leukocyte traffic. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like molecule that is thought to be a primary ligand for P-selectin. The interaction of P-selectin with PSGL-1 results in leukocyte rolling and recruitment of leukocytes to sites of inflammation and tissue injury. However, expression of PSGL-1 protein alone is insufficient for binding to P-selectin. Several posttranslational modifications of PSGL-1, including sialylation, sulfation, and fucosylation by alpha 1,3-fucosyltransferase(s) (FucT), are required for functional interaction with P-selectin. Recently, several groups have reported that PSGL-1 might also serve as a ligand for E-selectin. Differential posttranslational modifications of PSGL-1 may determine whether it can interact with either P- or E-selectin or both. To determine whether PSGL-1 is essential for adhesion to P- or E-selectin, we have constructed and analyzed a panel of stably transfected K562 cells. K562 cells express FucT-IV but not FucT-VII or PSGL-1, and do not bind to either E- or P-selectin. K562 cells transfected with PSGL-1 cDNA also did not bind to either P- or E-selectin. Binding to P-selectin occurred only when K562 cells were cotransfected with both FucT-VII and PSGL-1. In contrast, expression of FucT-VII alone was sufficient for E-selectin binding. These data demonstrate that expression of PSGL-1 is not required for adhesion of a stably transfected hematopoietic cell line to E-selectin, and suggest that FucT-IV alone cannot properly modify PSGL-1, expressed in transfected K562 cells, to bind P-selectin.