Glycobiology

Materials

Restriction enzymes were purchased from New England Biolabs, Boehringer Mannheim, or Promega. Taq polymerase was from Boehringer Mannheim, and Pfu polymerase was from Stratagene. [³²P]CTP, the Megaprime Labeling Kit, and prestained protein standard Rainbow markers were purchased from Amersham. The Superscript preamplification system was from Life Technologies. The polymerase chain reaction TA cloning kit, pZErO-1 vector, Zeocin, Pichia expression kit, and Pichia host strains GS115 and SMD1168 were purchased from Invitrogen. The Sephaglas band prep kit and the Ready to Go DNA labeling kit were purchased from Pharmacia. The T.cruzi epimastigote genomic [lambda]Fix II library was a gift from Dr. S.Teixeira and Dr. J.E.Donelson (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA). Plasmid purification columns, pQE-9 bacterial expression vector, and Ni-NTA resin were purchased from Qiagen. Swainsonine was from Boehringer Mannheim. Pichia culture medium components were purchased from Difco. Endoglycosidase H_f was from New England Biolabs. The DIG glycan detection kit, Endoglycosidase H, and protease inhibitor cocktail tablets were purchased from Boehringer Mannheim. PNGase F was a gift from Dr. J.Michael Pierce (University of Georgia, Athens, GA). The protein assay reagent and broad range protein standard markers were from Bio-Rad. The NEB Phototope kit was purchased from New England Biolabs. All other reagents were at least reagent grade and were obtained from standard suppliers.

PCR and subcloning of PCR product

Poly (A)+ RNA from Trypanosoma cruzi epimastigotes was reverse transcribed using random oligonucleotide primers and RNase H-Moloney murine leukemia virus reverse transcriptase (Superscript, BRL) (Moremen, 1989). Two degenerate oligonucleotide primers derived from areas of sequence similarity between the Dictyostelium lysosomal [alpha]-mannosidase and murine Golgi [alpha]-mannosidase (Merkle et al., 1997) were used in combination with a single-stranded T.cruzi cDNA template. The amplification of the T.cruzi [alpha]-mannosidase probe was performed as described previously (Liao et al., 1996; Merkle et al., 1997). The final reaction product was resolved on a 1% agarose gel containing ethidium bromide (0.5 µg/ml). Amplification products were purified from the gel using the Sephaglas DNA purification kit (Pharmacia) and subcloned into the pCR II vector (Invitrogen). Recombinant plasmids were isolated from liquid bacterial cultures using Qiagen columns (Qiagen Inc., Chatsworth, CA) and subjected to DNA sequencing. The 643 bp subcloned product was excised by digestion with EcoRI and purified after agarose electrophoresis as described above.

Genomic library screening

The 643 bp amplimer product from PCR amplification using degenerate primers was radiolabeled with [³²P]dCTP by random hexamer labeling using the Megaprime labeling system (Amersham), desalted, and used to screen a T.cruzi Tulahuén strain epimastigote genomic library in [lambda]Fix II. Plaque lifts were screened by standard formamide/SSC hybridization conditions (Sambrook et al., 1989) using Hybond-N filters (Amersham). Positive clones were plaque purified, and two of these clones were subjected to restriction analyses.

Subcloning into pZErO-1

DNA was isolated from plaque purified [lambda] clones (Qiagen) and digested with several restriction enzymes. Digestion products were separated on 0.5% agarose gels and transferred to Immobilon S neutral nylon membranes (Millipore) by capillary transfer using 10 × SSC. The 643 bp amplimer fragment was biotinylated as described by the manufacturer (NEB phototope kit, NEBiolabs) and used to probe Southern blots for potential full-length clones. Membranes were hybridized with the biotinylated amplimer and visualized as described by the manufacturer (NEB Phototope kit, New England Biolabs). Using other known [alpha]-mannosidase coding regions as a guide, the open reading frame was estimated to be approximately 3 kb in length, so fragments larger than 3 kb were chosen for subcloning.

A 4.2 kb SacI fragment from [lambda] clone #10 and a 4.9 kb SpeI-XhoI fragment from [lambda] clone #14 which hybridized to the amplimer probe were chosen for subcloning into the pZErO-1 vector. The restriction digestion products were separated on 0.5% agarose gels and bands, of the specified sizes were excised from the gel and purified using the Sephaglas DNA purification kit (Pharmacia). Fragments were subcloned into the pZErO-1 vector (Invitrogen) using complimentary sites and were designated TCM10 (4.2 kb SacI fragment) and TCM14 (4.9 kb SpeI-XhoI fragment). Recombinant plasmids were isolated from liquid bacterial cultures as described above and subjected to DNA sequencing.

DNA sequence analysis

T.cruzi [alpha]-mannosidase putative coding region fragments in plasmid vectors were sequenced using the dideoxy dye-terminator reaction (Sanger et al., 1977), Taq polymerase, and synthetic oligonucleotide primers and were analyzed on an Applied Biosystems 373A DNA sequencer (Molecular Genetics Instrumentation Facility, University of Georgia) using the standard protocol as described by the manufacturer. DNA sequence data were assembled into a contiguous sequence data base using the Sequencher program (Gene Codes Corporation, Ann Arbor, MI). Analysis of sequence similarities between DNA or protein sequences were performed using the Bestfit and Pileup programs of the University of Wisconsin Genetics Computer Group (GCG software, version 8.0).

Northern blot analysis

Total RNA was isolated from T.cruzi epimastigotes as described previously (Chirgwin et al., 1979). Ten or twenty-five micrograms of total epimastigote RNA were resolved on a 1% formaldehyde-agarose gel and transferred to Zeta-probe nylon membrane (Bio-Rad) as described previously (Lal et al., 1994). Blots were prehybridized, hybridized, and washed at 55°C, essentially as described previously (Moremen and Robbins, 1991) using a 1.8 kb radiolabeled probe (position #729-2674 of the coding region) generated by digestion of the subcloned coding region from TCM14 with MluI and PstI. The ³²P-labeled probe was generated using [³²P]dCTP (Amersham) and the Ready-To-Go labeling system (Pharmacia). Blots were visualized using a PhosphorImager (Molecular Dynamics).

Southern blot analysis

Genomic DNA from T.cruzi, T.brucei, and Leishmania strain CC-1 were isolated as described previously (Medina-Acosta and Cross, 1993). DNA samples (10 or 25 µg) were digested with the indicated restriction enzymes and the digestion products were separated on 1% agarose gels and transferred to Immobilon-S neutral nylon membranes (Millipore) by capillary blotting using 10× SSC. Membranes were hybridized with the biotinylated 1.8 kb MluI-PstI [alpha]-mannosidase probe and visualized as described above. A 1.2 kb BamHI fragment from T.brucei GPI-PLC (Mensa-Wilmot et al., 1995) and a 1.4 kb BamHI fragment from Leishmania amazonensis NAGT (Liu and Chang, 1992) were also biotinylated and used as control probes on Southern blots. Hybridizations were carried out at 65°C (high stringency), 55°C (medium stringency), or 42°C (low stringency) and chemiluminescent detection was performed as described by the manufacturer. Membranes were exposed on Hyperfilm (Amersham) or BioMax MR film (Eastman Kodak Co.).

Transformation of Pichiapastoris

The 5[prime] end of the 2.8 kb coding region of the T.cruzi [alpha]-mannosidase was remodeled to include an EcoRI site 15 bp upstream of the ATG start codon using sense primers of the sequence 5[prime]-CCCGAATTCTGGCTCTATTGCAAC-3[prime] in combination with an antisense primer (5[prime]-CCCCTCGAGTTTGATATTTCCAAATCGTCTTCCAG-3[prime]), that mapped to position 1745 in the [alpha]-mannosidase coding region. The 1772 bp PCR amplimer corresponding to the 5[prime] end of the [alpha]-mannosidase coding region was digested with MluI and ligated with a 2736 bp MluI-BamHI fragment corresponding to the 3[prime] end of the [alpha]-mannosidase coding region in pCRII to create the complete coding region with an added EcoRI site 15 bp upstream of the start codon. The [alpha]-mannosidase coding region, including 5[prime]UTR (15 bp) and 3[prime]UTR (189 bp), was excised from pCR II by digestion with EcoRI and ligated into the EcoRI site of the Pichia expression vector, pPICZ (Invitrogen). The expression construct was electroporated into both GS115 and SMD1168(pep4) Pichia host strains following the procedure outlined in the Pichia expression manual (Invitrogen) using a GenePulser Electroporator (Bio-Rad). Ten micrograms of PmeI-linearized recombinant vector was used for each transformation. Negative control transformations using PmeI-linearized vector without the [alpha]-mannosidase coding sequence were also performed. Positive transformants were selected for homologous recombination of the vector-encoded Zeocin resistance gene by growth on media containing 100 µg/ml Zeocin (Invitrogen). Ten colonies were selected from each transformation for methanol induction (Cregg et al., 1985) of the recombinant protein expression as described previously (Liao et al., 1996; Merkle et al., 1997). After the induction period, cultures were centrifuged at 2800 × g for 15 min and the clarified media was assayed for secreted [alpha]-mannosidase activity.

Enzyme and protein assays

The T.cruzi [alpha]-mannosidase was assayed using either p-nitrophenyl-[alpha]-d-mannopyranoside (pNP-Man) or 4-methylumbelliferyl-[alpha]-d-mannopyranoside (4MU-Man) as substrates. Enzyme assays of crude culture medium using pNP-Man as substrate were performed as described previously (Liao et al., 1996) with the exception that the assay buffer was 0.1 M sodium citrate, pH 3.5. Enzyme assays using 4MU-Man consisted of 25 mM sodium citrate buffer (pH 3.5), 30 mM 4MU-Man, and 10 µl of culture medium in a total volume of 250 µl. The mixture was incubated at 37°C for the indicated times, and the reaction was terminated by the addition of 2 ml of 100 mM sodium carbonate. The released 4-methylumbelliferone was quantitated using a Hoefer DyNA Quant 200 Fluorimeter with a fixed excitation band pass (365 nm) and an emission bandpass filter (460 nm). One unit of enzyme activity is defined at the amount of enzyme that releases 1 µmol of 4-methylumbelliferone in 1 h at 37°C. For determination of the pH optimum, 25 mM McIlvaine citrate-phosphate buffers (McIlvaine, 1921) varying from pH 2.0 to 7.5 were used in place of sodium citrate. When used as an inhibitor of [alpha]-mannosidase activity, swainsonine was dissolved to a concentration of 1.44 mM and added into the reaction mixture at various final concentrations. Protein concentration was determined by the method of Bradford (Bradford, 1976), using the Bio-Rad protein assay reagent and bovine serum albumin as standard.

Substrate specificity and thin layer chromatography

Substrate specificity was determined using radio-labeled oligosaccharides (a gift from Dr. M.A.J. Ferguson, University of Dundee) of defined structures as previously reported for the purified natural sources enzyme (Oeltmann et al., 1994). Incubations were carried out using concentrated crude culture medium from a methanol-induced Pichia transformant in 25 mM sodium citrate buffer at pH 4.0 in a total volume of 20 µl for the times indicated. Reaction products were analyzed using aluminum-backed silica gel 60 HPTLC plates as previously described (Schneider et al., 1993). After air drying, the plates were sprayed with En³Hance (NEN-Dupont) and exposed against Kodak X-Omat XAR-5 film at -70°C.

Purification of Pichia-expressed T.cruzi[alpha]-mannosidase

A SMD1168(pep4) Pichia transformant expressing the highest level of [alpha]-mannosidase activity was used for inoculation of a 20 liter fermentor culture in BMGY medium. The culture was allowed to grow to saturation, then methanol was added to a final concentration of 0.5%, and the culture was supplemented daily with methanol to maintain a concentration of 0.5%. After 5 days of induction, the medium was clarified in a Sharples continuous centrifuge (model AS-16P, Warminster, PA) at 15,000 r.p.m. for 60 min. The medium was filtered using a 0.45 µm filter, and sodium azide was added to a final concentration of 0.05% to prevent bacterial growth. The clarified supernatant was concentrated to 1.5 l by ultrafiltration with a 30 kDa cut-off tangential flow membrane (Pellicon 2, Millipore) and washed twice with an equal volume of 10 mM sodium phosphate (phosphate buffer), pH 7.2. The filtrate was adjusted to 1 mM ZnSO₄ and subjected to heat treatment at 70°C for 1 h as described for the purification of human lysosomal [alpha]-mannosidase B (Emiliani et al., 1995). Following the heat treatment the enzyme sample was incubated at 4°C for 1.5 h and centrifuged at 17,000 × g for 10 min. Solid ammonium sulfate was added to the supernatant to a final concentration of 1 M and the solution was applied to a phenyl-Sepharose column (26 mm × 140 mm, Pharmacia) at a flow rate of 1 ml/min. The column was washed with 60 ml of phosphate buffer containing 1 M ammonium sulfate, and the [alpha]-mannosidase activity was eluted with a 900 ml decreasing step gradient of 1-0 M ammonium sulfate in sodium phosphate buffer, (1-0.4 M, 180 ml; 0.4 M for 120 ml; 0.4-0.2 M, 300 ml; 0.2-0 M, 120 ml; 0 M for 180 ml) at a flow rate of 1 ml/min. Eluted fractions containing the enzyme activity were pooled and concentrated on a YM-100 membrane (Amicon) with repeated concentration and dilution to equilibrate the enzyme solution in phosphate buffer. The concentrated sample was applied at a flow rate of 1 ml/min onto a Mono Q 5/5 column (Pharmacia). The column was washed with 20 ml of phosphate buffer, and enzyme activity was eluted with a step gradient of 0-1 M NaCl in phosphate buffer (0-0.5 M, 40 ml; 0.5 M for 5 ml; 0.5-1 M, 20 ml; 1 M for 20 ml) at a flow rate of 1 ml/min. Peak fractions of [alpha]-mannosidase activity were pooled and concentrated in a Centricon-10 (Amicon) concentrator with repeated concentration and dilution to equilibrate the enzyme preparation in phosphate buffer containing 150 mM NaCl. The equilibrated sample (1.3 ml) was applied onto a Superdex-200 gel filtration column (16 mm × 600 mm, Pharmacia) preequilibrated with 10 mM phosphate buffer, 150 mM NaCl and the sample was eluted with the same buffer. Fractions containing [alpha]-mannosidase activity were pooled.

Deglycosylation reactions

Heat- and SDS-denatured, purified recombinant T.cruzi [alpha]-mannosidase (20 µg) was subjected to deglycosylation with 4000 U of recombinant endoglycosidase H_f (New England Biolabs) for 10 h at 37°C, following the manufacturer's protocol or with PNGase F (gift from Dr. J. M. Pierce, University of Georgia) with the same protocol and buffers as the endoglycosidase H_f digestions except that Triton X-100 was added to the reaction after the heat denaturation step at a final concentration of 2% and 1.4 units of PNGase F were added per microgram of purified protein. Reactions were incubated at 37°C for 10 h. Indicated reactions were carried out in the presence of a protease inhibitor cocktail (Boehringer Mannheim).

Purified recombinant T.cruzi [alpha]-mannosidase (80 µg) was subjected to degylcosylation by recombinant endoglycosidase H (Boehringer Mannheim) in 50 mM sodium citrate (pH 5.5) without heat or detergent denaturation, essentially as described by the manufacturer, except that a 20-fold excess of enzyme (275 mU/mg protein) was added and the digestion was carried out at 37°C for 10 h. Aliquots of the deglycosylated material were applied to a calibrated Superdex-200 (10 mm × 300 mm, Pharmacia) column in phosphate buffer containing 150 mM NaCl at a flow rate of 0.25 ml/min.

SDS-PAGE, immunoblotting, and lectin detection of glycoprotein

SDS-PAGE was carried out by the method of Laemmli (Laemmli, 1970). Gels were stained with Coomassie R-250 or Silver stain (Bio-Rad). Proteins resolved on SDS-PAGE gels were blotted onto PVDF membranes (Immobilon P, Millipore) as described previously (Moremen et al., 1991). Lectin detection of glycosylated protein bands on the blot was carried out as described previously (Liao et al., 1996). Carboxypeptidase Y was used as a positive control for GNA lectin detection.

NH₂-terminal sequencing of recombinant T.cruzi[alpha]-mannosidase

Purified recombinant a-mannosidase (8 µg) or endoglycosidase H-digested recombinant [alpha]-mannosidase (9 µg) were subjected to SDS-PAGE, electroblotted onto a PVDF membrane, stained with Ponceau S and subjected to sequencing on an ABI model 494 protein sequencer (Molecular Genetics Instrumentation Facility, University of Georgia; Aebersold et al., 1987).

Amino acid sequencing of [alpha]-mannosidase purified from T.cruzi epimastigotes

The [alpha]-mannosidase was purified from epimastigotes as described previously (Swanson et al., 1992; Oeltmann et al., 1994). An aliquot (50 µg in 25 µl) of the enzyme purified through the S-200 gel filtration step was subjected to SDS-PAGE and transferred to nitrocellulose as described previously (Aebersold et al., 1987; Bischoff et al., 1990) and subjected to solid-phase trypsin digestions of the nitrocellulose-bound protein followed by HPLC separation and sequencing of the eluted peptides at the Harvard Microchemistry facility (Cambridge, MA).

Preparation of antibody against recombinant NH₂-terminal 58 kDa of the [alpha]-mannosidase coding region

A 1680 base pair DNA fragment containing approximately half of the T.cruzi [alpha]-mannosidase coding region (position 70-1750) was amplified with the 5[prime] end remodeled to contain a BamHI site (5[prime] primer: 5[prime]-CCCGGATCCAAGCACACCGTACATCTTGTCGCGCACACC-3[prime]) and the 3[prime] end remodeled to contain a HindIII site (3[prime] primer: 5[prime]-ACTCCGGGAGCACGAATTCAAGCCTGCATTTTCGAACCC-3[prime]), using TCM10 as template. The PCR product was subcloned into the pQE-9 vector (Qiagen) for expression as an NH₂-terminal histidine-tagged peptide in E.coli. The vector construct was transformed into the M15[pREP4] E.coli strain and protein expression was induced for 2 h with 2 mM IPTG. The recombinant expression product was purified from an 8 M guanidine HCl extract of the cells by chromatography over a (Ni⁺²) NTA-affinity column described by the manufacturer (Qiagen). The purified protein (500 µg) was emulsified in an equal volume of Freund[prime]s complete adjuvant and used to immunize a male New Zealand White rabbit. Booster immunizations, using 500 µg protein in Freund[prime]s incomplete adjuvant, were given in 3 week intervals. The antibody preparation was diluted 1:2000 for detection of the Pichia expressed [alpha]-mannosidase on Western blots, and an alkaline phosphatase conjugated, goat anti-rabbit IgG (Promega) was used at a 1:5000 dilution as the secondary antibody.

Preparation of antibodies against the recombinant [alpha]-mannosidase and immunoprecipitation of enzyme activity

The purified, recombinant [alpha]-mannosidase was subjected to nondenaturing endo H deglycosylation and applied to a calibrated Superdex 200 column as described above. Pooled active fractions (6 µg) were emulsified in an equal volume of Freund[prime]s complete adjuvant and used to immunize a male New Zealand White rabbit. At 3 week intervals additional booster immunizations were given with 8 µg of enzyme and Freund's incomplete adjuvant.

Immunoprecipitation studies were performed using either concentrated Pichia media containing recombinant enzyme or crude T.cruzi epimastigote cell lysates (Swanson et al., 1992). Incubations and washes were carried out as described previously (Liao et al., 1996).