Serum level of long noncoding RNA B3GALT5-AS1 as a diagnostic biomarker of colorectal cancer.

Aim: Long noncoding RNA (lncRNA) B3GALT5-AS1 has been reported as a biomarker for cancer monitoring. This research aims to identify serum long noncoding RNA B3GALT5-AS1 as a new biomarker for the diagnosis of colorectal cancer (CRC) and evaluate its clinical value. Materials & methods: Serum B3GALT5-AS1 expression levels were measured by quantitative real-time PCR. Results: The level of B3GALT5-AS1 in CRC patients was significantly lower than that of healthy patients (p < 0.0001). Further exploration validated that high serum B3GALT5-AS1 level was related to tumor node metastasis (TNM) stage (p = 0.008) and histological differentiation (p = 0.027). Compared with the healthy control group, AUCROC of serum B3GALT5-AS1 in the CRC group was 0.762 with 95% CI: 0.698-0.826 (p < 0.0001). Conclusion: B3GALT5-AS1 may be served as a diagnostic marker for distinguishing CRC patients from healthy people.

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.

Cytokine profiles in Tibetan macaques following α-1,3-galactosyltransferase-knockout pig liver xenotransplantation.

BACKGROUND: Pig-to-nonhuman primate orthotopic liver xenotransplantation is often accompanied by thrombocytopenia and coagulation disorders. Furthermore, the release of cytokines can trigger cascade reactions of coagulation and immune attacks within transplant recipients. To better elucidate the process of inflammation in liver xenograft recipients, we utilized a modified heterotopic auxiliary liver xenotransplantation model for xeno-immunological research. We studied the cytokine profiles and the relationship between cytokine levels and xenograft function after liver xenotransplantation. METHODS: Appropriate donor and recipient matches were screened using complement-dependent cytotoxicity assays. Donor liver grafts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human CD47 (GTKO/CD47) were transplanted into Tibetan macaques via two different heterotrophic auxiliary liver xenotransplantation procedures. The cytokine profiles, hepatic function, and coagulation parameters were monitored during the clinical course of xenotransplantation. RESULTS: Xenograft blood flow was stable in recipients after heterotopic auxiliary transplantation. A Doppler examination indicated that the blood flow speed was faster in the hepatic artery (HA) and hepatic vein (HV) of xenografts subjected to the modified Sur II (HA-abdominal aorta+HV-inferior vena cava) procedure than in those subjected to our previously reported Sur I (HA-splenic artery+HV-left renal vein) procedure. Tibetan macaques receiving liver xenografts did not exhibit severe coagulation disorders or immune rejection. Although the recipients did suffer from a rapid loss of platelets, this loss was mild. In blood samples dynamically collected after xenotransplantation (post-Tx), dramatic increases in the levels of monocyte chemoattractant protein 1, interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor, IL-6, and interferon gamma-induced protein 10 were observed at 1 hour post-Tx, even under immunosuppression. We further confirmed that the elevation in individual cytokine levels was correlated with the onset of graft damage. Finally, the release of cytokines might contribute to leukocyte infiltration in the xenografts. CONCLUSION: Here, we established a modified auxiliary liver xenotransplantation model resulting in near-normal hepatic function. Inflammatory cytokines might contribute to early damage in liver xenografts. Controlling the systemic inflammatory response of recipients might prevent early post-Tx graft dysfunction.

Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates.

BACKGROUND: Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. METHODS: Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. RESULTS: The pooled human AB serum contained 0.38 µg/ml anti-Neu5Gc IgG and 0.085 µg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. CONCLUSIONS: Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

Genetic variant of C1GalT1 contributes to the susceptibility to IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is a common form of primary glomerulonephritis characterized by diffuse glomerular mesangial IgA1 deposition that leads to mesangial proliferation and chronic glomerular inflammation. Analyses of serum IgA1 from IgAN patients revealed an abnormal galactosylation of the O-linked carbohydrate moieties of IgA that may be a result of altered activity of core 1 beta1,3-galactosyltransferase (C1GalT1). To evaluate the association between C1GalT1 single nucleotide polymorphisms (SNPs) and IgAN, we performed a case control study on cohorts from the Italian population. METHODS: We sequenced C1GalT1 coding and promoter regions in 284 IgAN patients and 210 healthy controls. The functional role of 3' untranslated region (3'UTR) SNPs was studied using electrophoretic mobility shift assays and real-time quantitative PCR. RESULTS: We analyzed 8 SNPs in the C1GalT1 gene: 5 SNPs were in the promoter region and 3 SNPs in the 3'UTR. The allele 1365G in the 3'UTR was significantly more frequent in IgAN patients than in healthy controls. CONCLUSION: The 1365G allele and 1365G/G genotype seem to confer susceptibility to IgAN.

The synonymous 903C>G mutation in the alpha 1,4-galactosyltransferase gene in a Chinese woman with habitual abortion: A case report.

RATIONALE: Habitual abortion is caused by complex and diverse factors, such as genetic factors, immune factors, endocrine factors, viruses, bacterial infections, and so on. Allogeneic antibodies, generated due to blood-group incompatibilities between a female and her fetus, are sometimes important for habitual abortion. PATIENT CONCERNS: A 26-year-old woman had undergone abortions 3 times in July 2015 (17 weeks pregnant), March 2017 (15 weeks of gestation) and February 2018 (16 weeks pregnant) before she came to the Reproductive Medicine Center of our hospital for prenatal examinations without pregnancy. DIAGNOSES: Unexplained habitual abortion. INTERVENTIONS: A series of serological tests and nucleotide sequence of 1,4-galactosyltransferase (A4GALT) gene were performed. OUTCOMES: The patient was the rare p phenotype in P1P blood system and the patient's habitual abortion was caused by anti-PP1P antibody which was generated naturally in persons with p phenotype. There was a mutation (903C>G, CCC>CCG) in the 3rd exon of A4GALT gene, which is likely a significant contributor to p phenotype. LESSONS: This is the first case of habitual abortion caused by p phenotype due to independent 903C>G homozygous mutation with no similar record reported before, which indicates that it is a new class of mutation that leads to p phenotype.

Glycosyltransferase levels in tumors metastatic to liver and in uninvolved liver tissue.

Elevated levels of three plasma glycosyltransferases were associated with neoplasia in cancer patients, notably those with tumor metastatic to liver. We examined levels of sialyltransferase, galactosyltransferase, and fucosyltransferase in metastatic tumor and apparently uninvolved host liver tissue in attempts to delineate possible sources of elevated plasma enzyme levels. Highest levels of fucosyltransferase activity were found associated with tumor tissue; in contrast, sialyltransferase and galactosyltransferase activity was often highest at the tumor-liver interface.

Serum galactosyltransferase as a prognostic marker in patients with solid tumors.

The serum level of galactosyltransferase was measured in a group of 218 patients with a variety of solid tumors and most with advanced disease. The pretreatment enzyme level showed little potential as a diagnostic tumor marker, and its change with treatment did not reflect the initial response. There was, however, a significant correlation between the length of survival and the pretreatment enzyme level. Patients with normal levels survived over twice as long as those with elevated levels. When Cox's proportional hazards regression analysis was used to compare the prognostic potential of galactosyltransferase with a number of known clinical indicators of prognosis, the variable most related to survival was performance status (P less than 10(-4) followed by galactosyltransferase (P = 0.01) and then the extent of disease (P = 0.03). The other variables, such as previous therapy, the type, site, and size of primary tumor, did not contribute significantly to the relationship with survival. The pretreatment level of galactosyltransferase is therefore a relatively independent prognosticator of survival and, as such, could be potentially useful in patient management by increasing the accuracy of the initial assessment of prognosis.

Immunoassay of serum galactosyltransferase isoenzyme II in cancer patients and control subjects.

Serum galactosyltransferase isoenzyme II (GT-II) was assayed in 409 coded serum samples obtained from the National Cancer Institute Tumor Serum Bank using a monoclonal antibody-based immunoassay. The serum panel consisted of samples from patients with confirmed, metastatic ovarian, breast, stomach, esophageal, pancreatic, lung, colorectal, bladder, prostate, and cervical cancer, as well as benign disease controls corresponding to each cancer type, and confirmed healthy normal controls. The serum panel was matched for age and sex; 176 of 179 cancer patients had metastatic disease, and many had undergone previous therapy. GT-II was significantly elevated (P less than 0.01) in all pairwise tests (Wilcoxon) comparing cancer cases with normals and cancer cases with benign disease cases of the same site. A cutpoint of 200 milliunits of GT-II activity/ml of serum was selected, and only one of 50 normal control sera was elevated above this value, yielding a specificity of 98%. The overall sensitivity of the GT-II assay was 55.3%, with higher sensitivity shown by pancreatic (77%), prostate (65%), esophageal (64%), cervical (59%), and bladder cancer (58%).

Alterations in serum glycosyltransferases and 5'-nucleotidase in breast cancer patients.

We have measured sialyltransferase, galactosyltransferase, and fucosyltransferase as sell as 5'-nucleotidase in the serum of breast cancer patients. Serum sialyltransferase values in 65 normal healthy females ranged from 2.6 to 8.5 units, with a mean of 5.4. In 25 women with operable primary breast cancer, serum sialyltransferase levels were found to be between 6.2 and 15.4 units. Marked elevation of this enzyme level (range, 8.8 to 36 units) was observed in 48 patients with metastatic breast cancer. Galactosyltransferase and fucosyltransferase measurements, however, showed considerable overlap between the controls and the cancer patients. On the other hand serum 5'-nucleotidase and sialyltransferase in breast cancer patients showed very similar patterns. Thus, serum 5'-nucleotidase values in 44 normal females ranged from 11.4 to 23.2 units, whereas the levels found in 30 patients with metastasis were between 25 and 71.8 units. The tissue origin of abnormal levels of serum glycosyltransferases and 5'-nucleotidase was discussed in relation to their physiological significance as well as their role as markers for diagnosing early malignant breast neoplasm and for monitoring the extent of metastasis.

Clinical value of serum glycoprotein galactosyltransferase levels in different histological types of ovarian carcinoma.

Serum glycoprotein galactosyltransferase levels were determined in 28 healthy women and 113 patients with ovarian carcinoma with various histological types, at different clinical stages. Ovomucoid, which possesses terminal N-acetylglucosaminyl residues, was used as glycoprotein acceptor. Clinical correlations between galactosyltransferase levels and tumor burden were examined, as well as the variations due to histology. Follow-up studies could be done for 60 patients, and correlations with clinical evolution, established. Galactosyltransferase might be a promising marker for the diagnosis and follow-up of ovarian carcinomas.

Elevated thymic and serum galactosyltransferase with low-molecular-weight acceptor activity in murine leukemia.

Galactosyltransferase activity (UDP-galactose:N-acetyl-D-glucosamine-D-galactosyltransferase) could be measured in thymus and sera from different strains of mice. Total thymic homogenates or thymocyte preparations obtained from thymoma carrying AKR/J mice exhibited higher enzyme activity compared to nonleukemic control mice. A similar difference was also noted in Swiss mouse thymus which develop thymic leukemia upon a single injection of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide. Galactosidase, which was 25 times less active than galactosyltransferase, was not responsible for this difference. These observations were extended to an evaluation of the serum level of the enzyme as a potential tumor biomarker. A 3- to 4-fold increase in the activity of galactosyltransferase was detected in serum samples obtained from both leukemic mice models (AKR/J and Swiss) compared to the controls, whereas the sera from P388 tumor-bearing DBA/2 mice showed a statistically nonsignificant increase of only 20%. The data indicate that serum galactosyltransferase (that accepts the low-molecular-weight acceptor, N-acetyl-D-glucosamine) levels are elevated in the presence of thymic leukemia, and suggest the possibility of shedding of this enzyme from the tumor cells to the systemic circulation of the host. The implications, including the potential diagnostic significance of the results, are discussed.

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.

Elevation of 4 beta-galactosyltransferase activity for paragloboside synthesis in sera of patients with cancer.

Galactosyltransferase activities in sera of cancer patients were determined by assaying the formation of paragloboside from UDP-galactose and lactotriaosylceramide immobilized on microtiter plates by means of the enzyme-linked immunosorbent assay using a monoclonal antibody, H-11, directed to paragloboside. Enzyme properties were as follows. Optimum pH was 6.8 in cacodylate buffer, and Km values were 2 microM for lactotriaosylceramide and 29 microM for UDP-galactose. The enzyme activity was inhibited by the addition of alpha-lactalbumin. Glucose (20 mM) inhibited the enzyme activity in the presence of alpha-lactalbumin (0.1 mg/ml) but not in its absence. These enzyme properties are similar to those of bovine milk galactosyltransferase, indicating that the enzyme in the sera might be lactose synthetase. The enzyme activities in sera from patients with cancer, patients with benign disease, or a reference sample group were assayed. The activity was below the limit of detection (5.5 pmol/25 microliters serum/2 h) in the reference sample group. Remarkable elevations of the enzyme activity were observed with high incidence in patients with cancer, especially those with blood cancer (100%). A high incidence was observed in the progressive stage, and the enzyme activity was detected at stage 1 in lung, esophagus, stomach, colorectal, and testis cancer. The enzyme activity in sera from patients with benign disease was elevated in 22% of the patients. After effective therapies, the enzyme activity decreased to below the limit of detection. Release of the galactosyltransferase into culture medium of cancer cells could be demonstrated. These observations suggest that the galactosyltransferase is released from cancer tissue into the circulation. The present method for the assay of galactosyltransferase may be useful for the detection of patients with cancer and for monitoring neoplastic recurrence after therapy.

Mouse monoclonal antibodies which recognize a human (beta 1-4)galactosyl-transferase associated with tumor in body fluids.

Mouse monoclonal antibodies against human (beta 1-4)galactosyl-transferase (GalT) purified from human ovarian tumor effusion fluids were prepared and characterized. GalT purified from normal human plasma showed a single diffused band in nondenaturing polyacrylamide gel electrophoresis, but GalT purified from human ovarian tumor effusion fluids showed several oligomeric bands and a monomeric band in nondenaturing polyacrylamide gel electrophoresis. These oligomeric bands were dissociated into monomer by urea treatment and polymerized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies (MAb) were prepared by immunization of purified GalT from human ovarian tumor effusion fluids and classified into three groups. Type I MAbs (MAb8611, MAb8913, and MAb8919) reacted only to the GalT monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) reacted to both the GalT monomer and the GalT polymer. Type III MAbs (MAb7907, MAb8513, and MAb8677) reacted only to the GalT polymer. These MAbs except MAb7907 could recover GalT enzyme activity from effusion fluids by immunoprecipitation. A fraction passed through MAb8513 affinity chromatography still showed reactivity to MAb8919, demonstrating that an epitope of MAb8513 resides on a minor part of GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was developed, and serum samples from ovarian cancer patients and benign ovarian patients were tested. The levels of sandwich immunoassay of serum samples from cancer were elevated significantly compared to those from benign and did not necessarily correlate to total GalT enzyme activity in serum samples. These results suggested that MAb8513 (Type III) might recognize a unique GalT associated with tumor (GAT).

Identification of novel plasma glycosylation-associated markers of aging.

The pro- or anti-inflammatory activities of immunoglobulins G (IgGs) are controlled by the structure of the glycan N-linked to Asn297 of their heavy chain. The age-associated low grade inflammation (inflammaging) is associated with increased plasmatic levels of agalactosylated IgGs terminating with N-acetylglucosamine (IgG-G0) whose biogenesis has not been fully explained. Although the biosynthesis of glycans is in general mediated by glycosyltransferases associated with internal cell membranes, the extracellular glycosylation of circulating glycoproteins mediated by plasmatic glycosyltransferases has been recently demonstrated. In this study we have investigated the relationship between plasmatic glycosyltransferases, IgG glycosylation and inflammatory and aging markers. In cohorts of individuals ranging from infancy to centenarians we determined the activity of plasmatic ß4 galactosyltransferase(s) (B4GALTs) and of α2,6-sialyltransferase ST6GAL1, the glycosylation of IgG, the GlycoAge test (a glycosylation-based marker of aging) and the plasma level of inflammatory and liver damage markers. Our results show that: 1) plasmatic B4GALTs activity is a new marker of aging, showing a linear increase throughout the whole age range. 2) plasmatic ST6GAL1 was high only in children and in people above 80, showing a quadratic relationship with age. 3) Neither plasmatic glycosyltransferase correlated with markers of liver damage. 4) plasmatic ST6GAL1 showed a positive association with acute phase proteins in offspring of short lived parents, but not in centenarians or in their offspring. 5) Although the glycosylation of IgGs was not correlated with the level of the two plasmatic glycosyltransferases, it showed progressive age-associated changes consistent with a shift toward a pro-inflammatory glycotype.

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars. I. Determination of the degradation rate during the glycosyltransferase assays.

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phosphatases; 3, chemical decomposition of UDP-D-[14C]Gal; UDP-D-[14C]Glc and UDP-D-[14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn2+, Mg2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. These results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site.

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.

Identification of the product formed by human erythrocyte galactosyltransferase.

Sepharose 4B-immobilized desialylated ovine submaxillary mucin was used as an acceptor for galactose transfer from UDP-galactose, catalyzed by a Triton X-100-solubilized galactosyltransferase from human erythrocyte ghosts. The product could be cleaved from the insoluble acceptor substrate by alkaline borohydride treatment and identified on Bio-Gel P-2 as a disaccharide. The nature of the glycosidic bond of the isolated material was elucidated by periodate oxidation/NaB[3H]4 reduction/acid hydrolysis and subsequent identification of the aminopolyol formed as L-threosaminitol. Specific cleavage of the enzymatic product by beta-galactosidase indicated a beta-configuration for incorporated galactose. These data permit classification of the enzyme as UDP-galactose: alpha-D-N-acetylgalactosaminyl-protein beta (1 leads to 3) transferase. Furthermore, in the presence of Triton X-100, the enzyme from normal erythrocytes catalyzed transfer of galactose to the glycan moieties of asialo-agalacto-glycophorin in Tn-erythrocytes from a patient with permanent mixed-field polyagglutinability.