2Department of Cardiovascular-Thoracic Surgery, Rush University, 1653 West Congress Parkway, Chicago IL, 60612, USA, and 3Department of Immunology-Microbiology, Rush University Chicago IL, 60612, USA
Received on December 1, 2000; revised on March 22, 2001; accepted on March 22, 2001.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: recombinant 1,3galactosyltransferase/Pichia pastoris/anti-Gal/tumor vaccines/
-gal epitope
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We hypothesized that repeated immunization with autologous tumor cells or cell membranes expressing -gal epitopes will result in in vivo binding of anti-Gal IgG molecules to these epitopes (Galili and LaTemple, 1997
). The bound IgG molecules target the vaccinating tumor membranes to antigen-presenting cells (APCs), such as dendritic cells and macrophages, as a result of the binding of the Fc portion of anti-Gal on the membranes to Fc
receptors on APCs. These APCs transport the vaccinating tumor membranes to the adjacent lymph nodes, process the tumor-associated antigens (TAAs) within the tumor cell membranes, and present the TAA peptides in association with major histocompatibility complex molecules. This process, in turn, results in effective activation of anti-tumor T cells within the lymph nodes. The activated T cells can leave the lymph node, circulate in the body, and detect and destroy metastatic tumor cells expressing TAA. Recently, we demonstrated the efficacy of such tumor vaccines in protecting
1,3GT knock-out mice against the syngeneic melanoma tumor B16-BL6 (LaTemple et al., 1999
).
Processing human tumor cells from leukemia or lymphoma patients or tumor membranes, obtained from solid tumors, to express -gal epitopes may be performed in vitro by incubation with recombinant (r)
1,3GT and UDP-Gal (sugar donor), according to the following reaction:
The number of exposed N-acetyllactosaminyl residues, functioning as the sugar acceptor on cell surface carbohydrate chains, may be further increased by incubation with neuraminidase, an enzyme that removes sialic acid (SA) from the terminal structure SA2-3(6)Galß1-4GlcNAc-R (Galili and LaTemple, 1997; LaTemple et al., 1996
).
A major biochemical challenge in developing this novel approach for human tumor vaccine is the production of very large amounts of pure soluble r1,3GT that will suffice for enzymatic treatment of large amounts of tumor cells (> 1 x 109 cells) obtained from leukemia and lymphoma patients or tumor membranes (> 1g) obtained from solid tumors that are removed from cancer patients. No studies have demonstrated the synthesis of carbohydrate epitopes on such large amounts of cell membranes by recombinant glycosyltransferases. Moreover, synthesis of carbohydrate epitopes on cell membranes by full-length glycosyltransferases containing the transmembrane domain may require the use of detergents that maintain the solubility of the enzyme (Basu and Basu, 1973
; Blake and Goldstein, 1981
; Betteridge and Watkins, 1983
; Joziasse et al., 1990
). Such detergents are likely to be detrimental to the integrity of the vaccinating membranes.
Previous studies have shown that solubility of glycosyltransferases may be achieved by truncation of the trans-membrane and cytoplasmic domain without affecting the catalytic activity (Colley et al., 1989). For obtaining a soluble r
1,3GT, we have previously cloned a truncated cDNA corresponding to amino acids 61376 of the New World monkey (marmoset)
1,3GT (Henion et al., 1994
). The produced enzyme is soluble because it lacks the trans-membrane and cytoplasmic domains. We studied the possible use of the yeast Pichia pastoris for production and secretion of relatively large amounts of r
1,3GT into the culture medium. Yeast culture media lack proteins and endotoxin, thus greatly decreasing the risk of such contaminants in the vaccine.
The experience in producing recombinant glycosyltransferases in yeast is very limited. The only studies describing production of mammalian glycosyltransferases in P. pastoris have been those of Gallet et al. (1998) describing production of
1,3fucosyltransferase and of Malissard et al. (2000)
, who produced soluble human ß1,4galactosyltransferase,
2,6sialyltransferase and
1,3fucosyltransferase. Both studies used a methanol inducible promoter for production of the secreted enzymes. Recombinant
1,3GT could not be effectively produced in this inducible expression system. However, production of this enzyme in P. pastoris was found to be effective under a constitutive promoter. By using r
1,3GT produced in this system, we succeeded in synthesizing a large number of
-gal epitopes on bulk amounts of freshly obtained human acute myeloid leukemia cells and on membranes prepared from the tumor of an ovarian carcinoma patient.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The purity of the isolated enzymes was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). As shown in Figure 1, r1,3GT displayed two distinct protein bands with small differences in their mobility, corresponding to 40 and 41 kD, respectively. The (His)6 tag was found to be in the enzyme molecules comprising the two bands, as indicated in a western blot stained with anti-His antibody (Figure 1). These bands may result from two sizes of carbohydrate chains on r
1,3GT produced by the yeast. This enzyme has one N-glycosylation site at Asn 321 (Henion et al., 1994
). The presence of a carbohydrate chain linked to it is evident from its size, larger than the 37 kDa predicted from the 336-amino-acid protein chain that includes the (His)6 tag. It may be possible that the lower band reflects a partial degradation of approximately 10 amino acids. As measured by the colorimetric BCA reagent assay (Pierce), 2 mg of r
1,3GT could be purified from 1 L yeast culture.
|
|
As shown in Figure 3A, the maximal number of -gal epitopes on 1 x 108 human red cells was synthesized by 15 µg/ml of the enzyme and corresponded to a total of 5 x 1013 epitopes. This is implied from the finding that human red cells incubated with the enzyme at this concentration expressed fourfold less
-gal epitopes than rabbit red cells, that is, the amount of treated human red cells required for 50% inhibition of M86 binding was fourfold higher than that of rabbit red cells. Because rabbit red cells express 2 x 106
-gal epitopes per cell, the maximal number of
-gal epitopes synthesized de novo on neuraminidase-treated human red cells is approximately 5 x 105 epitopes per cell, that is, a total of 5 x 1013 epitopes on 1 x 108 human red cells per reaction. This number of epitopes is the maximal number of epitopes that can be synthesized on the human red cells because a threefold higher concentration of the enzyme (i.e., 50 µg/ml) yielded the same number of
-gal epitopes on the cell membranes (Figure 3A).
|
|
As many as 4 x 109 leukocytes containing > 90% AML blasts were obtained from the patient. The cells, resuspended at a concentration of 1 x 108cells/ml in enzyme buffer, maintained their intactness in this solution during incubation with r1,3GT and did not undergo lysis. Synthesis of
-gal epitopes was measured in cells incubated for 2 h at 37°C with r
1,3GT (50 µg/ml) in the presence or absence of neuraminidase (1 mU/ml). The synthesis of
-gal epitopes on the AML cells was first analyzed by ELISA with the peroxidase-conjugated lectin Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin), which interacts specifically with
-gal epitopes (Wood et al., 1979
). Original AML cells and neuraminidase-treated AML cells, incubated for 2 h with UDP-Gal, washed, and dried in ELISA wells, failed to bind BS lectin (Figure 4), implying the lack of endogenous synthesis of
-gal epitopes on these cells. Cells incubated with r
1,3GT expressed
-gal epitopes as indicated by the binding of the lectin. This was the result of the presence of a certain amount of naturally uncapped N-acetyllactosaminyl residues on the leukemia cells, functioning as sugar acceptors for the enzyme. Addition of neuraminidase to the cell suspension resulted in the removal of SA from cell surface glycoconjugates and the exposure of many penultimate N-acetyllactosalminyl residues, thus increasing the number of
-gal epitopes synthesized by r
1,3GT. This was indicated by the eightfold increase in BS lectin binding to the cells (Figure 4).
|
|
|
|
-gal epitopes were readily synthesized on membranes incubated for 2 h only with r
1,3GT (Figure 8). However, the number of these epitopes doubled on membranes incubated with r
1,3GT and neuraminidase. No
-gal epitopes were detectable on tumor membranes treated only with neuraminidase or on untreated membranes. Because the difference in BS lectin binding to membranes treated only with r
1,3GT and to membranes treated with both neuraminidase and r
1,3GT is smaller than that observed in AML cells, it is probable that the proportion of naturally uncapped N-acetyllactosaminyl residues on ovarian carcinoma membranes is higher than that on AML cells. Nevertheless, in both tumors, addition of neuraminidase results in a marked increase in synthesis of
-gal epitopes because of the increase in the number of sugar acceptors for r
1,3GT.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous attempts to produce large amounts of truncated marmoset r1,3GT in other expression systems proved to be inadequate for preparation of human tumor vaccines. Most (> 97%) of the r
1,3GT produced in bacteria was found to reside within inclusion bodies in an insoluble form (Galili and Anaraki, 1995
). Attempts to solubilize the recombinant enzyme within the inclusion bodies (e.g., by guanidinium chloride) resulted in its inactivation. In addition to the relatively low amounts of soluble r
1,3GT isolated from bacterial lysates, the possible contamination with bacterial endotoxin makes this enzyme unsuitable for tumor vaccines. The enzyme was subsequently produced in the mosquito cell line sF9 infected with baculovirus containing the
1,3GT gene (Henion et al., 1997
). Because the enzyme in this system is isolated from a cell lysate, there is the concern that the treated patient may develop hypersensitivity/allergic reaction to minute amounts of mosquito proteins that are not detectable by standard analytical methods and that may adhere to the vaccinating tumor membranes. In contrast, the medium used for yeast growth, the source for the secreted enzyme, contains only small peptides but no proteins. Therefore, the enzyme tagged with (His)6 that is isolated from yeast culture supernatant on nickel-Sepharose column is free of contaminating proteins. Moreover, because the r
1,3GT produced in P. pastoris does not involve lysis of cells or bacteria, its purification is much easier than the previously reported isolation from bacterial or cell lysates (Galili and Anaraki, 1995
; Henion et al., 1997
).
Recombinant 1,3GT was not effectively produced in P. pastoris under a methanol-inducible promoter PAOX (not shown). This was the result of low expression and partial inactivation of the enzyme, which may be caused by the methanol in the medium. In contrast, the enzyme was effectively produced in P. pastoris under constitutive expression of the PGAP promoter, resulting in the isolation of 2 mg r
1,3GT per L culture. This amount is similar to that reported for several other recombinant proteins produced in P. pastoris (Guo et al., 1995
; Weiss et al., 1995
). The enzyme could not be isolated directly from the culture medium, possibly because of histidine-containing peptides within the medium, which compete with the (His)6 tag of the enzyme for binding to the nickel-Sepharose column. However, after the enzyme was precipitated by high salt concentration and then dissolved in PBS containing 10 mM imidazole, it readily bound to the nickel-Sepharose column. The enzyme eluted by 250 mM imidazole from these columns was found to be very pure, as indicated by the lack of other bands in SDSPAGE gels. Analysis of the Km of this enzyme (not shown) was found to be similar to that of the same enzyme we have previously produced in mosquito sF9 cells (Henion et al., 1997
). The isolated enzyme was found to be highly stable at 4°C and in a frozen form, in the presence of glycerol as stabilizer. However, repeated thawing and freezing resulted in loss of activity (not shown).
The r1,3GT produced by yeast is most suitable for synthesizing
-gal epitopes on carbohydrate chains of tumor cells or cell membranes. Our data imply that incubation of as much as 200 mg/ml tumor membranes or 1 x 108 cells/ml with 1 mU/ml neuraminidase, 50 µg/ml r
1,3GT, and 1 mM UDP-Gal for 2 h at 37°C, represents optimal conditions for producing the maximal number of
-gal epitopes on the membranes. The recombinant enzyme is effectively removed from the treated membranes by the repeated washes and the use of 1 mM ethylenediaminetetraacetic acid (EDTA) in the washing buffer. Thus, autologous tumor vaccines that will be used in humans are likely to contain only the autologous membranes expressing
-gal epitopes and no contaminants.
This method of -gal epitope expression on vaccinating tumor membranes can be used with any type of human tumor membranes, because all human cells have carbohydrate chains with the terminal structure SA-Galß1-4GlcNAc-R. Future studies on the vaccination of cancer patients with autologous tumor vaccines expressing
-gal epitopes will determine whether the autologous tumor vaccines effectively induce stimulation of the immune system in humans, achieved by anti-Gal-mediated targeting of such vaccines to APCs. It may be possible that in some of the patients, the immune response to such vaccines will be effective enough to induce immune-mediated elimination of residual tumor cells that survive chemotherapy and irradiation treatments, and thus prevent the relapse of the disease.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Medium
The medium used was YPD, including the following materials: 1% yeast exract (Fisher Biotech, NJ), 2% peptone (Beckton Dickinson, MD), and 2% glucose (Sigma).
Tumor cells and cell membranes
Four billion leukemia cells were received from an AML patient in relapse who was treated for the removal of the tumor cells by leukophoresis. An ovarian carcinoma tissue ( 10 g) was received from a patient undergoing surgery for the removal of the tumor. Both patients were treated at Rush PresbyterianSt. Luke Medical Center (Chicago, IL), and the tumor materials were obtained under informed consent.
Construction of vectors for expression of 1,3GT in P. pastoris
The truncated portion of the New World monkey (marmoset) 1,3GT cDNA (Henion et al., 1994
) containing the catalytic domain and part of the stem region but lacking the trans-membrane and cytoplasmic domains, was inserted into the expression vector pGAPZ
A. For this purpose, the region coding for amino acids 61376 in the cDNA was amplified by polymerase chain reaction (PCR) with addition of BamHI site at the 5'-end and HindIII site at the 3'-end. The resulting PCR products were digested with BamHI and HindIII and cloned into the pQE-32 vector (Qiagen) at the BamHI and HindIII sites. The cloned
1,3GT gene sequence was found to be in frame at the 5'-end with the upstream (His)6 coding sequence. Digestion of the resulting vector with EcoRI and HindIII produced a DNA fragment containing
1,3GT gene and its upstream (His)6 tag coding sequence. This (His)6 tag attached to the enzyme is needed for affinity isolation of the enzyme by nickel-Sepharose columns. Subsequently, the HindIII site was blunted by filled-in reaction with Klenow fragment of DNA polymerase. This EcoRIHindIIIklenow fragment was subcloned into EcoRI and NotIklenow sites of pGAPZ
A. This vector containing a zeocin resistance gene and
1,3GT cDNA was linearized with AvrII and transformed into the yeast P. pastoris strain GS115 by electroporation, according to the manufactures protocol. The transformants with stable integration of the
1,3GT gene were selected in presence of 1 mg/ml zeocin.
Isolation of P. pastoris clones producing 1,3GT
Fifty individual clones grown in presence of zeocin were screened for production of the enzyme. The assay was previously described (LaTemple et al., 1996), and was based on the ability of the enzyme to transfer galactose from the sugar donor UDP-Gal to terminal N-acetyllactosaminyl residues on N-linked carbohydrate chains of asialofetuin. Fetuin is a protein obtained from fetal calf serum that carries three N-linked carbohydrate chains, on which the terminal LacNAc residues are capped by SA (Green et al., 1988
). The removal of these SA units exposes nine LacNAc residues that function as sugar acceptors. Synthesized
-gal epitope were identified by the subsequent binding of the monoclonal anti-Gal antibody M86 (Galili et al., 1998
), as measured by ELISA. Desialylation of fetuin was performed by incubation of fetuin in 50 mM sulfuric acid at 80°C for 2 h and subsequent repeated dialysis. Asialofetuin (20 µg/ml) in carbonate buffer pH 9.5 was used to coat microtiter ELISA plates. After blocking with 1% BSA in carbonate buffer the enzyme preparation at different dilutions in 100 mM 2-[N-morpholino]ethan sulfonate (MES) buffer (pH 6.2) containing 25 mM MnCl2 and 1 mM UDP-Gal were added to the plate. After incubation at 37°C for 1 h, the plates were washed and the M86 monoclonal anti-Gal antibody was added and incubated at room temperature for additional 1 h. Subsequently, the plates were washed and incubated with HRP-conjugated anti-mouse IgM as secondary antibodies for 1 h at room temperature. After additional washing the color reaction with O-phenylenediamine was measured at 492 nm.
Purification of r1,3 GT
The secreted r1,3GT in 1 L supernatants from 3-day cultures was precipitated with high salt (50% ammonium sulfate) and spun at 30,000 x g for 30 min. The pellet was resuspended in 20 ml PBS containing 10 mM imidazole, and the resulting solution was passed through a 1-ml nickel-Sepharose column (Invitrogen) that was equilibrated with PBS/10 mM imidazole. After extensive wash with PBS containing 25 mM imidazole, the bound enzyme was eluted with MES buffer containing 250 mM imidazole. The concentration of the enzyme was measured by the colorimetric reaction (BCA system, Pierce) and the purity of the enzyme was determined by SDSPAGE and western blot assay.
Analysis of purified r1,3GT by SDSPAGE and western blots
Affinity-purified 1,3GT (5 µg) was electrophoresed in 12% SDSPAGE and then stained with Coomassie blue. For western blot analysis, 1 µg of purified r
1,3GT was electrophoresed on SDSPAGE. The proteins were transferred to polyvinylidene difluoride membranes by semi-dry electroblotting. The membrane was blocked overnight at 4°C in PBS with 3% defatted milk. The membrane was first incubated with anti-His antibody that binds to the (His)6 tag, then with HRP-conjugated anti-mouse IgG. Color reaction was developed with diaminobenzidin (Sigma) substrate.
Analysis of synthesized -gal epitopes on fetuin by SDSPAGE and western blots
Fetuin (1 mg/ml) in MES buffer, pH 6.0, containing 25 mM MnCl2 and 1 mM UDP-Gal, was incubated in three 0.1-ml aliquots with either neuraminidase (Sigma), or r1,3GT (50 µg/ml) and neuraminidase (1 mU/ml). Subsequently, 8 µg of fetuin from each aliquot were subjected to SDSPAGE and Coomassie blue staining. Two micrograms of the same samples were subjected to western blot analysis by staining with human anti-Gal isolated from normal serum of blood type AB donors (Galili et al., 1984
, 1985, 1987), followed by HRPanti human IgG (Dako) or with the monoclonal anti-Gal M86 antibody (Galili et al., 1998
), followed by HRP-conjugated anti-mouse IgM as a secondary antibody.
Determining specific activity of r1,3GT with cell surface glycoconjugates as acceptors
Because the produced r1,3GT is to be used for the synthesis of
-gal epitopes on tumor cell membranes, the activity of the enzyme had to be determined with membrane-bound carbohydrate chains as acceptor, rather than with the disaccharide Galß1-4GlcNAC (LacNAc) in solution, which is usually used for assaying native and recombinant
1,3GT activity (Basu and Basu, 1973
; Blake and Goldstein, 1981
; Betteridge and Watkins, 1983
; Larsen et al., 1989
; Joziasse et al., 1990
; Henion et al., 1994
). This required the use of a novel assay designated ELISA inhibition assay, in which the total number of synthesized
-gal epitopes on membranes can be determined by the subsequent binding of the monoclonal anti-Gal antibody M86 (Galili et al., 1998
; Stone et al., 1998
; Tanemura et al., 2000
). This assay, which is analogous to radioimmunoassays, is a modification of the original assay for measuring
-gal epitope expression on glycoproteins by the use of the natural anti-Gal antibody (Thall and Galili, 1990
).
As a source of membranes expressing carbohydrate chains that function as sugar acceptors for r1,3GT, we used glutaraldehyde-fixed human red blood cells that were pretreated with neuraminidase (5 mU/ml). The red cells at a 10% concentration (i.e., 1 x 109 human red cells per ml) were mixed with r
1,3GT (50 µg/ml) in enzyme buffer (i.e.,100 mM MES buffer, pH 6.2, containing 25 mM MnCl2 and 1 mM UDP-Gal) in a total volume of 200 µl. The mixture was incubated at 37°C for 2 h. After three washes with saline, the red cells were subjected to serial twofold dilutions in 100-µl aliquots of PBS containing 1% BSA. The red cells in each dilution were mixed with equal volume of monoclonal anti-Gal M86 at the final dilution of 1:100 of the antibody, that is, a concentration of the antibody that yields ELISA absorption results at the slope of the binding curve. The mixture was incubated overnight at 4°C with constant rotation to enable maximum binding of the antibody to
-gal epitopes. Subsequently, the cells and bound antibodies were removed by centrifugation, and the activity of free M86 antibody remaining in the supernatant was determined by ELISA with
-gal BSA as solid phase antigen, using HRP-conjugated goat anti-mouse IgM antibody as secondary antibody.
Untreated human red cells do not bind the antibody because they completely lack -gal epitopes (Galili et al., 1987
). However, treated red cells that express de novo
-gal epitopes bind the antibody proportionally to the number of
-gal epitopes expressed on the cells. Therefore, the amount of free M86 antibody remaining in the supernatant is inversely proportional to the number of epitopes per cell. By comparing the binding of M86 to cells with known number of
-gal epitopes per cell (standard cells) to that of the antibody binding to the treated human red cells, it is possible to determine the number of
-gal epitopes synthesized de novo on the human red cells (Galili et al., 1998
). Rabbit red cells were used as the standard cells because they were previously shown to express
2 x 106
-gal epitopes per cell (Galili et al., 1998
). Thus, if the treated human red cells are 10-fold less effective than rabbit red cells in the ELISA inhibition assay (i.e., 10-fold higher concentration of human red cells than that of rabbit red cells is required for 50% inhibition of anti-Gal M86 binding), the number of
-gal epitopes on the tested red cells is approximately 2 x 105 per red cell. Quantification of
-gal epitope expression on leukemia cells treated with r
1,3GT was determined by the same ELISA inhibition assay.
Synthesis of -gal epitopes on human leukemia cells and ovarian carcinoma membranes
A total of 4 x 109 cells AML cells were received from a patient undergoing leukophoresis treatment. The cells were brought to a concentration of 1 x 108 cells/ml in the enzyme buffer that includes saline, 25 mM MES (pH 6.2), 25 mM MnCl2, and 1 mM UDP-Gal. Ten milliliters of this cell suspension were incubated with r1,3GT, (50 µg/ml), and neuraminidase (1 mU/ml) for 2 h at 37°C. Cells treated only with neuraminidase or only with
1,3GT served as controls. At the end of incubation the cells were washed twice with PBS containing 1 mM EDTA and twice with PBS. For demonstration
-gal epitope synthesis, the cells at 2 x 106 cells/ml were plated into ELISA wells as 50 µl per well. After the cells were dried in these microtiter wells, they strongly adhered to the wells. The plates were blocked with PBS containing 1% BSA. The
-gal epitopes on the cells were detected by the subsequent binding of HRP-conjugated BS lectin at various concentrations of the lectin. After additional washing the color reaction with O-phenylenediamine was measured at 492 nm, according to a previously described assay (LaTemple et al., 1996
).
Similar -gal synthesis was performed with ovarian carcinoma tumor membrane homogenate at 200 mg/ml in the enzyme buffer. After completion of the
-gal epitope synthesis the membranes were washed, plated as 2 mg/ml in ELISA wells, and dried. The epitopes were subsequently detected by BS-lectin as described above for leukemia cells.
Flow cytometry analysis of -gal epitope expression on AML cells
AML cells were incubated at a concentration of 1 x 106 cells/ml for 2 h at 4°C with 10 µg/ml FITCBS lectin in PBS containing 1% BSA. Cells were then washed three times with PBS, fixed, and analyzed in a FACS Calibur flow cytometer (Becton Dickinson).
Western blot analysis of -gal epitopes expression on leukemia cells
Twenty micrograms of enzymatically treated or control leukemia cell membranes were subjected to SDSPAGE, blotted, and immunostained with human anti-Gal followed by HRPanti human IgG as described above for immunostaining of fetuin on western blots.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Betteridge, A., and Watkins, W.M. (1983) Two -3-D galactosyltransferases in rabbit stomach mucosa with different acceptor substrate specificities. Eur. J. Biochem., 132, 2935.[Abstract]
Blake, D.D., and Goldstein, I.J. (1981) An -D-galactosyltransferase in Ehrlich ascites tumor cells: Biosynthesis and characterization of a trisaccharide (
-D-galacto(1-3)-N-acetyllactosamine). J. Biol. Chem., 256, 53875393.
Blanken, W.M., and van den Eijnden, D.H. (1985) Biosynthesis of terminal Gal1-3Gal81-4GlcNAc-R oligosaccharide sequence on glycoconjugates: Purification and acceptor specificity of a UDP-Gal: N-acetyllactosamine
1, 3galactosyltransferase. J. Biol. Chem., 260, 1297212934.
Colley, K.J., Lee, E.U., Adler, B., Browne, J.K., and Paulson, J.C. (1989) Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide. J. Biol. Chem., 264, 1761917622.
Collins, B.H., Cotterell, A.H., McCurry, K.R., Alvarado, C.G., Magee, J.C., Parker, W., and Platt, J.L. (1994) Cardiac xenografts between primate species provide evidence of the -galactosyl determinant in hyperacute rejection. J. Immunol., 154, 55005510.
Galili, U. (1993) Interaction of the natural anti-Gal antibody with -galactosyl epitopes: A major obstacle for xenotransplantation in humans. Immunol. Today, 14, 480482.[ISI][Medline]
Galili, U., and Anaraki, F. (1995) Synthesis of the -galactosyl (Gal
1-3Gal-ß1-4GlcNAc-R) epitope on humans cells by recombinant primate
1,3galactosyltransferase expressed in E. coli. Glycobiology, 5, 783791.[Abstract]
Galili, U., and LaTemple, D.C. (1997) Natural anti-Gal antibody as a universal augmenter of autologous tumor vaccine immunogenicity. Immunol. Today, 18, 281285.[ISI][Medline]
Galili, U., and Swanson, K. (1991) Gene sequences suggest inactivation of 1,3 galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proc. Natl Acad. Sci. USA, 88, 74017404.[Abstract]
Galili, U., Rachmilewitz, E.A., Peleg, A., and Flechner, I. (1984) A unique natural human IgG antibody with anti--galactosyl specificity. J. Exp. Med., 160, 15191531.[Abstract]
Galili, U., Macher, B.A., Buehler, J., and Shohet, S.B. (1985) Human natural anti--galactosyl IgG. II. The specific recognition of
(1-3)-linked galactose residues. J. Exp. Med., 162, 573582.[Abstract]
Galili, U., Clark, M.R., Shohet, S.B., Buehler, J., and Macher, B.A. (1987) Evolutionary relationship between the anti-Gal antibody and the Gal1-3Gal epitope in primates. Proc. Natl Acad. Sci. USA, 84, 13691373.[Abstract]
Galili, U., Shohet, S.B., Kobrin, E., Stults, C.L.M., and Macher, B.A. (1988) Man, apes, and Old World monkeys differ from other mammals in the expression of -galactosyl epitopes on nucleated cells. J. Biol. Chem., 263, 1775517762.
Galili, U., LaTemple, D.C., and Radic, M.Z. (1998) A sensitive assay for measuring -gal epitope expression on cells by a monoclonal anti-Gal antibody. Transplantation, 65, 11291132.[ISI][Medline]
Gallet, P.F., Vaujour, H., Petit, J.M., Maftah, A., Oulmouden, A., Oriol, R., Le Narvor, C., Guilloton, M., and Julien, R. (1998) Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris. Glycobiology, 8, 919925.
Good, A.H., Cooper, D.C.K., Malcolm, A.J., Ippolito, R.M., Koren, E., Neethling, F.A., Ye, Y., Zuhdi, N., and Lamontage, L.R. (1992) Identification of carbohydrate structures which bind human anti-porcine antibodies: implication for discordant xenografting in man. Transplant Proc., 24, 559562.[ISI][Medline]
Green, E.D., Adelt, G., Baenziger, J.U., Wilson, S., and Van Halbeek, H. (1988) The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. J. Biol. Chem., 263, 1825318268.
Guo, W., Gonzalez-Candelas, L., and Kolattukudy, P.E. (1995) Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. J. Bacteriol., 177, 70707077.[Abstract]
Henion, T.R., Macher, B.A., Anaraki, F., and Galili, U. (1994) Defining the minimal size of catalytically active primate 1, 3 galactosyltransferase: structure function studies on the recombinant truncated enzyme. Glycobiology, 4, 193201.[Abstract]
Henion, T.R., Gerhard, W., Anaraki, F., and Galili, U. (1997) Synthesis of -gal epitopes on influenza virus vaccines by recombinant
1,3galactosyltransferase enables the formation of immune complexes with the natural anti-Gal antibody. Vaccine, 15, 11741182.[ISI][Medline]
Joziasse, D.H., Shaper, J.H., van den Eijnden, D.H., Van Tunen, A.H., and Shaper, N.L. (1989) Bovine 1-3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J. Biol. Chem., 264, 1429014297.
Joziasse, D.H., Shaper, N.L., Salyer, L.S., van den Eijnden, D.H., van der Spoel, A.C., and Shaper, J.H. (1990) 1-3-Galactosyltransferase: the use of recombinant enzyme for the synthesis of
-galactosylated glycoconjugates. Eur. J. Biochem., 191, 7583.[Abstract]
Larsen, R.D., Rajan, V.P., Ruff, M., Kukowska-Latallo, J., Cummings, R.D., and Lowe, J.B. (1989) Isolation of a cDNA encoding murine UDP galactose: ßD-galactosyl-1,4-N-acetyl-D-glucosaminide 1,3-galactosyltransferase: Expression cloning by gene transfer. Proc. Natl Acad. Sci. USA, 86, 82278231.[Abstract]
Larsen, R.D., Rivera-Marrero, C.A., Ernst, L.K., Cummings, R.D., and Lowe, J.B. (1990) Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal -D-Galß(1,4)-D-GlcNAc
(1,3)galactosyltransferase cDNA. J. Biol. Chem., 265, 70557062.
LaTemple, D.C., Henion, T.R., Anaraki, F., and Galili, U. (1996) Synthesis of -galactosyl epitopes by recombinant
1,3galactosyltransferase for opsonization of human tumor cell vaccines by anti-galactose. Cancer Res., 56, 30693074.[Abstract]
LaTemple, D.C., Abrams, J.T., Zhang, S.U., and Galili, U. (1999) Increased immunogenicity of tumor vaccines complexed with anti-Gal: Studies in knock out mice for 1,3galactosyltransferase. Cancer Res., 59, 34173423.
Malissard, M., Zeng, S., and Berger, E.G. (2000) Expression of functional soluble forms of human ß-1,4-galactosyltransferase I, -2,6-sialyltransferase, and
-1,3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris. Biochem. Biophys. Res. Commun., 267, 16973.[ISI][Medline]
Sandrin, M., Vaughan, H.A., Dabkowski, P.L., and McKenzie, I.F.C. (1993) Anti-pig IgM antibodies in human serum react predominantly with Gal 1-3Gal epitopes. Proc. Natl Acad. Sci. USA, 90, 1139111395.[Abstract]
Smith, D.F., Larsen, R.D., Mattox, S., Lowe, J.B., and Cummings, R.D. (1990) Transfer and expression of a murine UDP-Gal: -D-Gal
1, 3-galactosyltransferase gene in transfected Chinese Hamster Ovary cells. Competition reactions between the
1,3-galactosyltransferase and the endogenous
2,3-sialyltransferase, J. Biol. Chem., 265, 62256234.
Stone, K.R., Ayala, G., Goldstein, J., Hurst, R., Walgenbach, A., and Galili, U. (1998) Porcine cartilage transplants in cynomolgus monkey: III. Transplantation of -galactosidase treated porcine cartilage. Transplantation, 65, 15771583.[ISI][Medline]
Tanemura, M., Maruyama, S., and Galili, U. (2000) Differential expression of -gal epitopes (Gal
1-3Galß1-4GlcNAc-R) on pig and mouse organs. Transplantation, 69, 187190.[ISI][Medline]
Thall, A., and Galili, U. (1990) The differential expression of Gal 1
3Galß1
4GlcNAc-R residues on mammalian secreted N-glycosylated glycoproteins. Biochemistry, 29, 39593965.[ISI][Medline]
Weiss, H.M., Haase, W., Michel, H., and Reilander, H. (1995) Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization. FEBS Lett., 377, 451456.[Medline]
Wood, C., Kabat, E.A., Murphy, L.A., and Goldstein, I.J. (1979) Inmunochemical studies on the combining sites of two isolectins A4 and B4 isolated from Bandeiraea simplicifolia. Arch. Biochem. Biophys., 198, 18.[ISI][Medline]