Glycosylation using GalNac-T4 transferase

In this example, GalNAc-T4 was expressed in Sf9 insect cells. Expression constructs designed to contain amino acid residues 32-578 of the coding sequence of the putative GalNAc-T4 gene shown in SEQ ID NO:1 prepared by genomic PCR of two clones containing GalNAc-T4 (described in Bennett et al. (1998), supra) were cloned into a BamHI site of the expression vector pAcGP67 (Pharmingen), and the expression construct was sequenced. The constructs pAcGP67-GalNAc-T4 506V sol and pAcGP67-GalNAc-T4 506 I sol, were designed to yield a putative soluble form of the GalNAc-T4 protein with an N-terminal end positioned immediately C-terminal to a potential transmembrane domain and including the entire sequence expected to contain the catalytic domain. Control constructs pAcGP67-GalNAc-T1-sol, pAcGP67-GalNAc-T2-sol, pAcGP67-GalNAc-T3-sol, and pAcGP67-O 2 -sol were prepared as previously described in White et al., J. Biol. Chem 270:24156 (1995); Bennett et al., J. Biol. Chem. 271:17006 (1996); and Bennett et al., Biochem. Biophys. Res. Comm. 211:347 (1995).

A full coding expression construct was prepared by PCR and cloned into BamHI sites of the expression vector pVL1392 (Pharmingen). Co-transfection of Sf9 cells with pAcGP67-constructs or pVL-constructs and Baculo-Gold™ DNA was performed according to the manufacturer's specifications. Briefly, 0.4 μg construct was mixed with 0.1 μg Baculo-Gold DNA and co-transfected in Sf9 cells in 24-well plates. Ninety-six hours post-transfection, recombinant virus was amplified in 6-well plates at dilutions of 1:10 and 1:50. Titer of amplified virus was estimated by titration in 24-well plates with monitoring of GalNAc-transferase activities. Initial transferase assays were performed on supernatants of Sf9 cells in 6-well plates infected first or second amplified virus titers representing end-point dilutions giving optimal enzyme activities. Transferase assays of the full coding expression construct were performed by extracting washed cells in 1% Triton X-100 as described in Sorenson et al., J. Biol. Chem. 270:24166 (1995).

In this example, secreted GalNAc-T4 was stably expressed in Chinese Hamster Ovary cells (CHO cells). A truncated construct designed to contain amino acids 32-578 of the coding sequence of GalNAc-T4 (shown in SEQ ID NO:1 was prepared by genomic PCR using primer pairs EBHC314 (5′-AGCGGATCCGGTCAAGAAGGCTCTCAGACCTC-3′, SEQ ID NO:4) and EBHC307 (5′-AGCGGATCCGACGAAAGTGCTGTTGTGCTC-3′, SEQ ID NO:5). Bennett et al.(1998), supra. The product was cloned into a modified pcDNA3 vector (Invitrogen).

The pcDNA3 vector was modified to include 19 amino acids of the gamma-interferon signal sequence by directional insertion of a synthetic sequence of 91 bp coding for the interferon sequence with KpnI and BamHI flanking sites. The modified pcDNA3 vector was constructed as follows. Four synthetic oligonucleotides were synthesized: INFFOR (5′-cggggtaccggaaacgatgaaatatacaag-3′, SEQ ID NO:6); INFREVA (5′-ggcggatccaggcagatcacagccaagagaacccaaaacg-3′, SEQ ID NO:7); INFREVB (5′-gcggatcccaggcagatcacagccaagagaacccaaaacg-3′, SEQ ID NO:8); and INFREVC (5′-gcggatccccaggcagatcacagccaagagaacccaaaacg3′, SEQ ID NO:9). Oligonucleotide primer pairs INFFOR/INFREVA, INFFOR/INFREVB and INFFOR/INFREVC were used to PCR amplify an interferon coding DNA fragment from human genomic DNA under the following conditions: 95° C. for 30 seconds, 60° C. for 5 seconds, 72° C. for 15 seconds, using Ampli-Taq (Perkin-Elmer Cetus, Conn.) and a model 480 Thermocycler (Perkin-Elmer). See FIG. 1 . The use of three 3′ primers spaced one base apart yields three vectors with a BamHI site positioned for any of three reading frames with respect to the signal sequence.

The GalNAc-T4 construct was cloned into pcDNA3-INF-B, and correct insertion was confirmed by sequencing. The predicted coding region of the construct is shown in FIG. 1 and SEQ ID NO:10.

CHO-K1 cells (ATCC) were transfected using 0.2 μg DNA and 5 μg lipofectamine (Invitrogen) in subconfluent 6 well plates according to the manufacturer's protocol. After 48 hours, the medium was changed and 400 μg/ml G418 was added. At 72 hours 10-20% of the wells were trypsinized and the percentage of cells expressing GalNAc-T4 was evaluated by immunocytology using an anti-GalNAc-T4 monoclonal antibody, UH6.

UH6 was prepared by immunizing mice with a purified GalNAc-T4 preparation that gave a single band of approximately 58,000 Daltons on SDS-PAGE Coomassie-stained gel. Balb/c mice were immunized with one subcutaneous or intraperitoneal injection of 10 ul undenatured protein in Freund's complete adjuvant, followed by two injections with Freund's incomplete adjuvant, and finally an intravenous booster without adjuvant. Eyebleeds were taken 7 days after third immunization, and the titer and specificity of anti-GalNAc-transferase antibodies was evaluated. Fusion to NS-1 and the cloning procedure was as described in White et al., Biochemistry 29:2740 (1990). The monoclonal antibody UH6 was selected for reactivity with unfixed cells and/or tissues, as well as ability to immunoprecipitate GalNAc-T4 activity. Hybridomas were selected by three criteria: i) differential reactivity in ELISA assays with purified recombinant enzymes; ii) immunocytology on Sf9 cells two days after infection with Baculovirus containing GalNAc-transferases, GalNAc-T1, -T2 or -T3; and iii) differential immunoprecipitation of active recombinant enzymes.

ELISA analysis was performed as described by White et al. (1990), supra, using purified recombinant GalNAc-T1, -T2, -T3, -T4 and AOSM, using an initial antigen concentration of 10 μg/ml.

The immunocytology assay involved washing trypsinized cells twice in PBS and airdrying the washed cells onto coverslides. Dried slides were fixed in 100% icecold acetone for 10 min, dried, and incubated with monoclonal anti-GalNAc-T4 antibody for 1 hour. After washing with PBS, slides were incubated with FITC-conjugated rabbit anti-mouse IG for 30 minutes, washed with PBS and mounted in glycerol and analyzed by microscopy.

Immunoprecipitation of recombinant human GalNAc-transferase was performed as follows. Secreted forms of human GalNAc-transferases were expressed in Sf9 cells, and media were harvested three days post-infection and used as enzyme source. Protein G Sepharose was saturated sequentially with rabbit anti-mouse IgG and monoclonal antibodies as culture supernatants. A 5% suspension of Protein G beads was added to Sf9 medium containing either GalNAc-T1, -T2, -T3 or -T4. After incubation for 1 hour at 4 degrees C, beads were washed in PBS, and resuspended in 25 mM Tris (pH 7.4), 0.25% Triton X-100. GalNAc-transferase activities were measured in the supernatants and the washed pellets using a standard reaction mixture containing 25 mM Tris (pH 7.4), 10 mM MnCl 2 , 0.25% Triton X-100, 100 μM UDP-[ 14 C]-GalNAc (2,000 cpm/nmol), and 25 μg acceptor peptide substrate. UH6 selectively immunoprecipitated GalNAc-T4 activity but not GalNAc-T1, -T2 or -T3.

Western blot analysis with purified recombinant enzymes was also performed, but it proved difficult to select antibodies reactive with both the native and the denatured GalNAc-T4 enzyme. The antibody UH6 is therefore likely to be directed to a conformational epitope, and detects the native conformation of GalNAc-T4.

Based on the frequency of positive cells the residual transfectant cells from Example 2 were trypsinized and plated in 96 well plates. Two rounds of screening and cloning by limiting dilution using immunoreactivity with UH6 were performed and clones reaching over 50% positive cells were selected, and tested for level of secreted enzyme in supernatant of confluent cultures. The intensity of immunoreactivity by the cytology assay correlated in all cases with level of GalNAc-T4 enzyme activity found in spent media from clones.

Expression of pAcGP67-GalNAc-T4 506V sol, as described in Example 1, resulted in GalNAc-transferase activity in the culture medium of infected cells that was greater than background values obtained with uninfected controls or cells infected with the histo-blood group O 2 gene. Table I shows the substrate specificities of purified recombinant GalNAc-T4 and GalNAc-T2. Activities measured with the mucin derived substrates, Muc7, EA2, and Muc2 (see Table I for structures and SEQ ID NOs:19, 25 and 18, respectively), were only 2-7 fold over background values. Very low activities with a few other substrates were also observed, but for many of the “mucin-like” substrates a relative high background with Sf9 cells infected with irrelevant constructs made assessment difficult. GalNAc-T4 was expressed in High Five cells and purified to near homogeneity, however, very low endogenous activity was detected with some substrates in the same fractions when medium from cells infected with irrelevant expression constructs were used in the same purification strategy. The activities presented in Table I were obtained with purified GalNAc-T4 and -T2, and are expressed as mU/mg (the concentration of enzyme protein estimated by SDS-PAGE using BSA as standard). The highest activities with GalNAc-T4 were found from assays of Sf9 medium with the mucin peptides Muc7, EA2, and Muc2, however, the efficiencies with these substrates were considerably lower than GalNAc-T2.

In this example, it was demonstrated that GalNAc-T4 complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat. As seen in Table I, GalNAc-T4 showed somewhat poor activities with the MUC1 derived peptides Muc-1a, Muc-1b, and TAP24. The glycopeptide, GalNAc 4 TAP24 (Table I and SEQ ID NO:17), was tested because it has all the acceptor sites for GalNAc-T1, -T2, and -T3 occupied, see Wandell et al., supra., and therefore is not a substrate for these enzymes. Surprisingly, a low but significant activity with this substrate was observed with purified GalNAc-T4 (Table I, Table II). Detailed analysis of the activity revealed a strikingly low apparent Km of 90 μM, although Vmax was low (Table II). Analysis of the reaction products by capillary zone electrophoresis, as described by Wandell et al., supra., indicated that a total of two moles of GalNAc were incorporated when the reaction was run to completion. Analysis of the HPLC purified product of the reaction by mass spectrometry confined the presence of six moles of GalNAc.

In this example, it was shown that GalNAc-T4 can O-glycosylate Thr 57 in PSGL-1. Two peptide designs of the N-terminal sequence of mature PSGL-1 were tested (Table I and SEQ ID NOs:20 and 20). In a recent study, Thr 57 of PSGL-1 was identified as the carrier site of an O-glycan required for P-selection binding. Liu et al., J. Biol. Chem. 273:7078 (1998). GalNAc-T1, -T2, and -T3, exhibited poor activities with both peptide designs, but HPLC analysis of prolonged reaction indicated that both Thr 44 and Thr 57 served as substrates, although the reaction did not go to completion for either site (Tables I and II). GalNAc-T4, in contrast, showed low activity with the PSGL-1b peptide containing both Thr 44 and Thr 57 , but not with PSGL-1a without the Thr 57 site. The reaction went to completion and the apparent Km for this peptide was very low (Tables I and II). Analysis of the exhaustively glycosylated PSGL-1b peptide by mass spectrometry confined that a single GalNAc was incorporated (data not shown). A strong substrate inhibition with the negatively charged PSGL-1b peptide was observed at concentrations greater than 1 mM. The Km of purified GalNAc-T4 for UDP-GalNAc was 160 μM using the Muc7 acceptor substrate. No incorporation with UDP-Gal or UDP-GlcNAc was found using the same peptide.

One clone from Example 2, designated CHO/GalNAc-T4/21A1, was selected and culture medium of confluent T-flasks contained up to 0.95 mU/ml activity measured with Muc7 in the standard assay. A stable CHO clone, CHO/GaNAc-T3/H3-6, secreting 5 mU/ml GalNAc-T3 (using Muc1a acceptor substrate) has also been established using the same immunoscreening procedure, using an anti-GalNAc-T3 monoclonal antibody. GalNAc-T3 has a specific activity of 0.5 U/mg with Mucla, and GalNAc-T4 appears to have a specific activity of 0.053 U/mg with Muc7 (Table I). Thus the protein secretion level of CHO/GalNAc-T4/21A1 is approximately 20mg/L, which is comparable to, or better than, that of CHO/GalNAc-T3/H3-6, which secretes approximately 10 mg/L. Since there is no detectable endogenously secreted GalNAc-transferase activity in the medium of wild-type CHO, this enzyme source should be very valuable for studies of substrates with low efficiency. Using the method of the invention, other human glycosyltransferases (β4Gal-T2, βGal-T5, the GaM2 βGalNAc-transferase, and α3FUT5) have been expressed in CHO cells and secretion levels of 10-30 mg/L obtained.

Expression of pAcGP67-GalNAc-T4 506 I sol did not result in detectable GalNAc-transferase activity in the culture medium of infected cells that was greater than background values obtain with uninfected controls or cells infected with irrelevant constructs. However, the activity detectable with pAcGP67-GalNAc-T4 506V sol directly in the culture medium of infected Sf9 cells was only a few fold over background. The expression of pAcGP67-GalNAc-T4 506I sol as well as pAcGP67-GalNAc-T4 507V sol and pVL-GalNAc-T4 516V full in Sf9 cells was monitored by immunocytological reaction with UH6. Cells infected with pAcGP67-GalNAcT4 506V sol were clearly positive, but the number of cells which were positive, and the intensity, was lower than for cells infected with pACGP67-GalNAc-T4 506V sol and PVL-GalNAc-T4 506V full. The expression of GalNAc-Tr 506V sol was equivalent (in terms of number of positive cells and intensity) to that previously reported for GalNAc-T1, -T2, and -T3 constructs using specific monoclonal antibodies to these enzymes. Expression of pAcGP67-GalNAc-T4 506I sol in High Five cells followed by purification produced a catalytically active enzyme fraction with activities essentially similar to that of the GalNAc-T4 506V sol variant.

Expression of pVL-GalNAc-T4 506V full in Sf9 cells did not produce significant GalNAc-transferase activities in homogenates with the same substrates as found for the secreted construct. However, this assay is influenced by a very high endogenous GalNAc-transferase background. The expression level was evaluated by immunocytology with UH6, and cells infected with the full coding construct stained stronger than those infected with the pAcGP67-GalNAc-T4 506V sol construct. When a Triton X-100 homogenate of infected Sf9 cells was incubated with GalNAc 4 TAP24 for 24 hours with one addition of UDP-GalNAc and extra enzyme, no incorporation was detected by MALDI-TOF of HPLC-purified product.

Each document referenced in the foregoing disclosure is incorporated by reference to the extent relevant to making or using the invention as claimed.

1

atggcggtga ggtggacttg ggcaggcaag acctgcctgc tgctggcgtt tttaacagtg 60

gcctatatct tcgtggagct cttggtctct acttttcatg cctccgcagg agccggccgt 120

gccagggagc tggggtcaag aaggctctca gacctccaga aaaatacgga ggatttgtct 180

cgaccgcttt ataagaagcc ccctgcagat tcccgtgcac ttggggagtg ggggaaagcc 240

agcaaactcc agctcaacga ggatgaactg aagcagcaag aagaactcat tgagagatac 300

gccatcaata tttacctcag tgacaggatt tccctgcatc gacacataga ggataaaaga 360

atgtatgagt gtaagtccca gaagttcaac tataggacac ttcctaccac ctctgttatc 420

attgctttct ataacgaagc ctggtcgact ttgctccgta ccattcacag tgttttagaa 480

acttctcctg cagttctttt gaaagagatc atcttggtgg atgacttgag tgacagagtt 540

tatttgaaga cacaacttga aacttacatc agcaatcttg atagagtacg cttgattagg 600

accaataagc gagaggggct ggttagggcc cgtctgattg gggccacttt cgccactggg 660

gacgtcctca ctttcctgta ttgtcactgt gagtgtaatt ccggttggct ggaaccgctt 720

ttggaaagga ttgggagata tgaaacagca gttgtgtgtc ctgttataga cacaattgat 780

tggaatactt ttgaattcta tatgcagata ggggagccca tgattggtgg gtttgactgg 840

cgtttaacat ttcagtggca ttctgtcccc aaacaggaaa gggacaggcg gatatcaaga 900

attgacccca tcagatcacc taccatggct ggaggactgt ttgctgtcag caagaaatat 960

tttcagtacc ttggaacgta tgacacagga atggaagtgt ggggaggtga aaaccttgag 1020

ctgtctttta gggtgtggca gtgtggtggc aaattggaga tccacccgtg ttcccacgtg 1080

ggccatgtgt tccccaagcg ggcaccatat gctcgcccca atttcctaca gaatactgct 1140

cgggcagcag aagtttggat ggatgaatac aaagagcact tctacaatag aaaccctcca 1200

gcaagaaaag aagcttatgg tgatatttct gaaagaaaat tactacgaga gcggttgaga 1260

tgcaagagct ttgactggta tttgaaaaac gtttttccta atttacatgt tccagaggat 1320

agaccaggct ggcatggggc tattcgcagt agagggatct cgtctgaatg tttagattat 1380

aattctcctg acaacaaccc cacaggtgct aacctttcac tgtttggatg ccatggtcaa 1440

ggaggcaatc aattctttga atatacttca aacaaagaaa taaggtttaa ttctgtgaca 1500

gagttatgtg cagaggtacc tgagcaaaaa aattatgtgg gaatgcaaaa ttgtcccaaa 1560

gatgggttcc ctgtaccagc aaacattatt tggcatttta aagaagatgg aactattttt 1620

cacccacact caggactgtg tcttagtgct tatcggacac cggagggccg acctgatgta 1680

caaatgagaa cttgtgatgc tctagataaa aatcaaattt ggagttttga gaaatag 1737

2

578