Defining the genetic control of human blood plasma N-glycome using genome-wide association study.

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.

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.

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.

CD15s is a potential biomarker of serious bacterial infection in infants admitted to hospital.

Early recognition of serious bacterial infection (SBI) in children is essential for better treatment outcome. Flow cytometry analysis of neutrophil surface molecules has been more frequently utilized as a tool for diagnosis of infection. The infants (n = 105) under 6 months of age presenting to the pediatric emergency department with fever without apparent source who were hospitalized with suspicion of having SBI were enrolled in this prospective study. Sixty-nine infants were included into the training pool and were classified into bacterial or viral infection group. Validation pool consisted of 36 infants. The values of white blood cells counts, absolute neutrophil count (ANC), C-reactive protein (CRP), procalcitonin (PCT), neutrophil CD11b, CD15s and CD64 expression, and the percentage (%CD15s+) and absolute count (AC-CD15s+) of CD15s+ neutrophils were determined. In infants with SBI, %CD15s+ was 10.5 times more likely to be higher than the cut-off value. ANC, CRP, PCT, CD64, and AC-CD15s+ were also found as useful biomarkers for differentiation between bacterial and viral infection. The best fit multivariate logistic regression model included CRP, PCT, and %CD15s+ as strong predictors of SBI. The model's sensitivity (87 %) and specificity (83 %) indicated high model's accuracy. After validation on independent dataset, model's accuracy maintained high: 86 % sensitivity and 93 % specificity, confirming its reliability and supporting CRP, PCT, and %CD15s+ as real predictors. The findings of this study support assumption made in the literature on significance of CD15s in inflammation processes. Also, this study demonstrated for the first time that CD15s is potentially valuable biomarker of SBI in infants.

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.

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).

Novel serum proteomic signatures in a non-human primate model of retinal injury.

PURPOSE: To identify candidate protein biomarkers in sera indicative of acute retinal injury. METHODS: We used laser photocoagulation as a model of acute retinal injury in Rhesus macaques. In a paired-control study design, we collected serum from each animal (n=6) at 4 h, 1 day, and 3 days following a mock procedure and then again following retinal laser treatment that produced mild lesions. Samples were fractionated by isoelectric focusing, digested with trypsin, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Spectral counting was used to determine relative protein abundances and identify proteins with statistically significant differences between control and treated sera. RESULTS: Mild retinal injury was confirmed by fundus photography and histological examination. The average number of total proteins detected by LC-MS/MS was 908±82 among samples from all three time points. Following statistical analysis and employing stringent filtering criteria, a total of 19 proteins were identified as being significantly more abundant in sera following laser-induced retinal injury, relative to control sera. Many of the proteins detected were unique to one time point. However, four proteins (phosphoglycerate kinase 1, keratin 18, Lewis alpha-3-fucosyltransferase, and ephrin receptor A2) showed differences that were significant at both 4 h and 1 day after laser treatment, followed by a decrease to baseline levels by day 3. CONCLUSIONS: A serum biomarker response to mild retinal laser injury was demonstrated in a primate model. Among the proteins detected with highest significant differences, most are upregulated within 24 h, and their appearance in the serum is transient. It is conceivable that a panel of these proteins could provide a means for detecting the acute-phase response to retinal injury. Further investigation of these candidate biomarkers and their correlation to retinal damage is warranted.

Decrease of human serum fucosyltransferase as an indicator of successful tumor therapy.

Surgical removal of colon carcinomas leads to a decrease in the rate of incorporation of [14C]fucose into its endogenous acceptor in human serum; normal incorporation rates are attained within 14 days. A similar time course has been determined for alpha2- and alpha3-fucosyltransferase when either desialo- or desialodegalactofetuin are employed as exogenous acceptors. A correlation has also been seen between transferase activity and the therapeutic response of patients with breast cancer. These results indicate that the determination of fucosyltransferase activity can facilitate the diagnosis of neoplasia, and the success of surgery, chemotherapy, or radiation.

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.

Electrofocusing patterns of fucosyltransferases in plasma of patients with neoplastic disease.

Electrofocusing patterns of plasma fucosyltransferases provide information concerning marrow status of patients with myeloproliferative disorders. Three enzymes were detected in normal plasmas using an acceptor terminating in the sequence N-acetylglucosamine-galactose. The enzyme which focused at pH 4.7 was elevated during rapid proliferation of myeloid cells, e.g., acute myelogenous leukemias and certain infectious diseases. Activity at pI = 5.1 was decreased in acute myelogenous leukemia patients, and from other observations, appears related to the level of erythropoietic activity. Acceptor studies show this enzyme to be specified by the H gene. A third enzyme focused at pH 5.5 and appeared to be correlated with a later step in granulocytes maturation. Two other plasma fucosyltransferases (pl = 5.6 and 8.3) were detected with a high-molecular-weight acceptor terminating in N-acetylglucosamine. This activity was markedly elevated during regeneration of a normal marrow population during drug-induced remission of acute myelogenous leukemia. Additional isoenzymes were detected, using this acceptor, in plasma of patients with certain solid tumors and multiple myeloma. However, the new isoelectric points observed (pH 6.0, 6.9, and 7.8) suggest these enzymes are probably not derived from hematopoietic tissues.

Electrofocusing patterns of fucosyltransferase activity in plasma of patients with chronic granulocytic leukemia.

Fucosyltransferase activity was measured in plasma samples obtained from patients with chronic granulocytic leukemia in different disease stages. Total enzyme activity was generally elevated in untreated and in stable disease; a marked further elevation was noted 30 to 60 days before development of blast crisis. Electrofocusing studies indicate the presence of three major enzymes. The level of enzyme which focused at pH 5.6 was elevated in untreated disease and rose markedly during blastic transformation. A second enzyme focused at pH 5.1 and appeared to be related to marrow erythroid turnover. A third enzyme with pI = 4.7 was elevated only during marrow regeneration which followed myelosuppressive therapy. These data suggest the enzyme with pI = 5.6 to be a specific marker in chronic granulocytic leukemia.

Significance of altered alpha-2-L-fucosyltransferase levels in serum of leukemic patients.

Serum alpha-2-L-fucosyltransferase levels were measured in 20 patients with leukemia. Lower than normal levels were found in patients with acute leukemia and higher than normal activity was demonstrated in patients with chronic granulocytic leukemia. alpha-2-L-Fucosyltransferase activity is expressed in platelets, and in both groups of leukemic patients this enzyme activity in serum was found to correlate directly with the platelet count. Assays on platelets from normal donors and from leukemic patients showed that the changes in level of the enzyme in serum result from changes in platelet numbers and not from alterations in the cellular expression of the transferase in leukemia. alpha-2-L-Fucosyltransferase levels in serum are therefore not per se diagnostic markers of leukemia.

Electrofocusing pattern of fucosyltransferase activity in human leukemic cells.

Fucosyltransferase (FT) activity of normal lymphocytes, normal granulocytes, and various types of human leukemic cells and electrofocusing pattern of FT activity in human leukemic cells and normal lymphocytes were examined using asialofetuin as an acceptor. Levels of FT activity in normal lymphocytes were higher than those of normal granulocytes in which FT activity was almost undetectable. The FT activity was higher in blast cells of acute myeloblastic leukemia and chronic myelogenous leukemia in blast crisis than in blast cells of acute lymphoblastic leukemia and the chronic phase of chronic myelogenous leukemia. The level of FT activity was lower in cells of chronic lymphocytic leukemia than that of normal lymphocytes, but it was higher than that of normal granulocytes. Three main isoelectric forms of FT in leukemic blast cells were identified by isoelectrofocusing, and they each had a characteristic focusing point: around pH 4.5 (peak 1); pH 4.9 (peak 2); and pH 5.2 (peak 3). In blast cells of myeloid leukemia, the activity of peak 3 was markedly higher than those of peaks 1 and 2. In blast cells of lymphoid leukemia, the activity of peak 3 was also the highest, but the activity of peak 2 was higher than that in myeloid blast cells. In normal lymphocytes, the major isoelectric form of FT was focused at around pH 4.9 and peak 3 was undetectable. These results indicated apparent differences not only in FT activity but also in isoelectric forms of FT between myeloid leukemic cells and lymphoid leukemic cells.

Leukemia-induced alterations of serum glycosyltransferase enzymes.

Studies on blood group A and H glycosyltransferase enzymes in 54 patients with acute myeloid leukemia were carried out on serum derived from blood samples taken prior to treatment, and in 16 cases, further tests were performed during clinical remission and at the time of relapse. The enzyme assay procedures, using low-molecular-weight compounds as sugar acceptors and radioactive nucleotide sugars as the donor substrates, have been described by Chester et al. (Eur. J. Biochem., 69:583, 1976). Abnormally low values of H enzyme (expressed as percentage of radioactive sugar incorporated into product; (that is, 1 to 3%) were observed in practically all presentation sera, but the values reverted to normal levels (3 to 15%) at the time of clinical remission and then became low once more with the development of drug resistance and clinical relapse. A enzyme levels measured in presentation sera which had demonstrated abnormal H enzyme were mostly within the normal range. In 2 of 5 A1 patients; sera and in all of three A2 patients increases in enzyme levels were observed in remission as compared with presentation serum samples. The depression of biosynthetic enzymes in acute leukemic sera could not be accounted for on the basis of competitive inhibitors or catabolic enzymes. It is proposed that changes of serum glycosyltransferase enzymes reflect alterations in a leukemic cell population and that knowledge of these changes may be of value in prognosis in acute leukemia.

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.

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.

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.