Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

INTRODUCTION

Lectins are a structurally heterogeneous group of carbohydrate-binding proteins comprising distinct families of evolutionarily related proteins (Peumans et al., 2001). The most thoroughly investigated lectins are those from plants, in particular the Leguminosae. Legume lectins are a large group of structurally similar proteins with distinct carbohydrate specificities. Concanavalin A (ConA), found in the seeds of Canavalia ensiformis, was the first lectin to be sequenced (Cunningham et al., 1975; Wang et al., 1975) and to have its three-dimensional structure determined by X-ray crystallography (Becker et al., 1975; Hardman and Ainsworth, 1976). The many biochemical, biophysical, and structural studies carried out in ConA make this protein the best-characterized plant lectin. Indeed, ConA is the prototype of legume lectins.

Protein splicing is a post-translational event, in which the translated protein undergoes cleavage, resulting in the loss of an internal spacer sequence, while the remaining flanking domains are rejoined via a peptide bond (Cooper and Stevens, 1993; Wallace, 1993). C. ensiformis lectin is synthesized by a unique mechanism in maturing seeds. Based on information from pulse-chase analysis, cDNA cloning, and amino acid sequencing of the processing intermediates, it is known that ConA undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed (Carrington et al., 1985; Herman et al., 1985; Bowles et al., 1986; Chrispeels et al., 1986; Faye and Chrispeels, 1987).

ConBr, the glucose/mannose-binding lectin found in C. brasiliensis seeds (Moreira and Cavada, 1984), has 99% amino acid sequence identity with ConA (Grangeiro et al., 1997). These lectins display the same primary carbohydrate binding specificity for residues of D-glucose, D-mannose and derivatives, and both proteins possess enhanced affinities for the branched chain trimannoside, 3,6-di-O-(a-D-mannopyranosyl)-D-mannose (Dam et al., 1998), which is found in the core region of all asparagine-linked (N-linked) carbohydrates. Furthermore, ConBr and ConA essentially recognize the same set of binding epitopes on the structure of the trimannoside (Dam et al., 2000). The amino acids at positions 58 (glycine in ConBr and aspartic acid in ConA) and 70 (glycine in ConBr and alanine in ConA) are the only residues that differ between ConA and ConBr (Grangeiro et al., 1997; Sanz-Aparicio et al., 1997). Based on a comparison of the amino acid sequence of the precursor of ConBr (prepro-ConBr), deduced from the cDNA, with the established amino acid sequence of the lectin mature subunit (Grangeiro et al., 1997), the same mechanism of protein splicing observed during ConA biosynthesis is also involved in the production of mature ConBr (Figure 1).

Heterologous expression of prepro-ConBr in the yeast Pichia pastoris would help to determine whether the lectin maturation machinery is specific to C. brasiliensis or is conserved in other eukaryotic cells. We report here an initial characterization of P. pastoris clones transformed with the coding sequence for prepro-ConBr.

MATERIAL AND METHODS

Cell strains, plasmids and cell cultures

P. pastoris strain GS115 cells and pPICZB expression plasmid were obtained from Invitrogen. Medium compounds were purchased from Amersham Biosciences and from Oxoid. All medium formulations and abbreviations are described in the EasySelection Pichia Expression Kit manual, version F (www.invitrogen.com). All the cells were stored in 10% (v/v) glycerol at -20°C.

Expression vector construction

A DNA fragment encoding prepro-ConBr was amplified by PCR using C. brasiliensis genomic DNA as a template. The oligonucleotide primers were 5’ (forward): 5’- GAATTCAAAAATGTCTGCCATCTCAAAGAAATC-3’ and 3’ (reverse): 5’-GCTCTAGAACCACGGTAGCAAT-3’. EcoRI (forward) and XbaI (reverse) sites were included at the 5’ end of each primer. Primer sequences were designed in such a way to place the open-reading frame for prepro-ConBr in frame with the C-terminal tag. In addition, the design of the forward primer placed the start codon ATG as part of a yeast consensus sequence (Romanos et al., 1992). Primer sequences were based on the published sequence of ConBr gene (Grangeiro et al., 1997), which is deposited in GenBank (accession number Y13904). The amplification was carried out in a 25-µL reaction volume containing 50 ng template DNA, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl₂, 200 mM of each dATP, dCTP, dGTP, and dTTP (Amersham Biosciences), 0.5 µM of each primer and 2.5 units of Taq DNA polymerase (Amersham Biosciences). PCR reactions were carried out in an MJ Research (Watertown, MD) PTC-200 thermocycler programmed for an initial denaturation (5 min at 95°C) followed by 30 cycles of 1 min at 94°C, 1 min at 45°C and 2 min at 72°C. PCR products were purified from 1% agarose gel slices and cloned into the pPICZB plasmid vector (Invitrogen), using the restriction sites incorporated into the primer sequences.

Pichia pastoris transformation and induction of expression of the recombinant lectin

The recombinant plasmid was linearized by restriction with SacI (SstI) and transformed into P. pastoris strain GS115 by electroporation. Transformants were selected on YPD-sorbitol plates containing 200 µg/mL zeocin (Invitrogen) and incubated at 30°C. The integration of prepro-ConBr coding sequence in the genome of P. pastoris was confirmed by PCR, using genomic DNA samples from 20 individual zeocin-resistant clones as templates and primers specific for the coding sequence, as described in the previous section. Ten clones were then screened for the expression of recombinant lectin on a small scale using flask cultures. A single colony from each clone was grown overnight in 5 mL MGYH medium containing 200 µg/mL zeocin, spun down, resuspended in sterile distilled water and inoculated in 25 mL MMH medium. The cultures were incubated at 30°C with continuous shaking (250 rpm) and fresh 100% methanol was added twice a day to a final concentration of 0.5% (v/v). Culture samples (1 mL) were taken at various stages (0, 24, 48, 72, 96, and 120 h) during cell growth in the presence of methanol, and immediately centrifuged (12,000 g for 10 min). The supernatants and cell pellets were frozen in liquid nitrogen and stored at -20°C, for later analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.

SDS-PAGE and Western blot analysis

SDS-PAGE was done as described by Laemmli (1970), using 15% slab gels. Western blot analysis (Towbin et al., 1979) was carried out in nitrocellulose membranes (Hybond^TM C Extra, Amersham Biosciences, USA) using rabbit anti-ConBr antibodies and goat anti-rabbit IgG conjugated to horseradish peroxidase (Calbiochem, USA). Rabbit antibodies directed against ConBr were prepared by immunization with native ConBr purified by affinity chromatography on Sephadex G-50, as previously described (Moreira and Cavada, 1984). Antibodies specific to ConBr were purified from the serum of immunized animals using a ConBr-Sepharose column. Western blotting procedures followed standard methods (Sambrook et al., 1989).

RESULTS AND DISCUSSION

A PCR product encoding the prepro-ConBr was amplified from C. brasiliensis genomic DNA and cloned into pPICZB, a plasmid vector designed for intracellular expression in P. pastoris. As shown in Figure 1, the amplified DNA fragment encodes a polypeptide with 290 amino acids (aa), comprising a 29-aa signal peptide, the C-terminal domain (aa 119-237) found in the mature lectin, a 15-aa linker peptide, the N-terminal domain (aa 1-118) of the native protein, and a 9-aa C-terminal extension (Grangeiro et al., 1997). Integration of prepro-ConBr coding sequence in the yeast genome was confirmed by PCR in 20 clones of P. pastoris selected in the presence of zeocin. This yielded a single 870-bp PCR product, which is the expected size of the prepro-ConBr coding sequence (data not shown).

Ten transformed colonies of P. pastoris were randomly chosen and checked for effective recombinant protein production. Both cells and supernatants from methanol-induced cultures were analyzed by Western blot for each clone. In this screening, clonal variation in the expression of the recombinant lectin was observed, with two of 10 clones showing no detectable expression of rConBr (data not shown). Clonal variation in heterologous expression of Phaseolus vulgaris lectin E in P. pastoris has also been observed (Baumgartner et al., 2002). Western blot analysis was made of the two clones with the highest ConBr expression level. It was evident that upon induction a protein with an apparent molecular mass of about 34.5-kDa, which reacted with anti-ConBr antibodies, was produced (Figure 2). This immunoreactive band was not detected in the cell culture just before induction. Production of this 34.5 kDa band was detected in the cells 48-72 h after induction with methanol, and it remained until 120 h (Figure 2). In addition, as early as 24 h after induction, multiple immunoreactive bands with higher molecular masses were also detected in the cells of both clones, and they lasted until 120 h (Figure 2). These diffuse bands or smears at higher molecular masses are diagnostic of protein glycosylation (Raemaekers et al., 1999). Indeed, prepro-ConBr contains a single-potential N-glycosylation site located in the 15-aa linker peptide (Figure 1). However, native ConBr purified from C. brasiliensis seeds is not glycosylated. In the case of the homologous lectin ConA, it has been shown that the oligosaccharide side chain is removed during the transport of the glycosylated precursor via the Golgi into the protein storage vesicles of cotyledonary cells (Herman et al., 1985; Faye and Chrispeels, 1987). N-linked oligosaccharide side chains found on glycloproteins expressed in Pichia are mainly of the high-mannose type, and most of them have 8-14 mannose residues; thus they would be expected to increase the molecular mass of a glycoprotein by approximately 2 kDa (Cregg et al., 1993). Consequently, the high-molecular mass bands that reacted with anti-ConBr antibodies appear to be hyperglycosylated unprocessed ConBr.

A single-, high-molecular mass band that cross-reacted with anti-ConBr antibodies was also detected in the supernatants of the induced cultures (Figure 2). This suggests that ConBr’s native signal peptide directed the secretion of a soluble form of the lectin.

In the pPICZB vector, the recombinant lectin is produced as a fusion protein containing C-terminal tags, the myc epitope and a polyhistidine (6xHis) sequence, which allow detection of the expressed protein and its purification by immobilized metal ion affinity chromatography, respectively. The myc epitope and the polyhistidine tag contribute 2.5 kDa to the size of the expressed protein (Invitrogen). In addition, most of the high-mannose oligosaccharide side chains present in Pichia glycoproteins contribute 2 kDa to the mass of the expressed polypeptide (Cregg et al., 1993). Taking these findings into account, the 34.5-kDa polypeptide that is recognized by anti-ConBr antibodies can be explained by the removal of both the native signal peptide as well as the single-N-linked oligosaccharide chain, when prepro-ConBr is expressed in P. pastoris. Therefore, the 34.5-kDa band would correspond to pro-ConBr fused with the C-terminal tags. On the other hand, SDS-PAGE of purified native ConBr has shown that the mature lectin consists of a major 30-kDa band and small proportions of 16- and 12-kDa fragments (Moreira and Cavada, 1984). N-terminal amino acid sequencing of electroblotted samples, as well as comparisons of the amino acid sequence of the precursor of ConBr (prepro-ConBr) deduced from the cDNA with the known amino acid sequence of the lectin mature subunit (Grangeiro et al., 1997), indicated that ConBr precursor is post-translationally processed by a protein-splicing mechanism. In this mechanism, the precursor of ConBr is cleaved into two distinct chains that are religated by transpeptidation to form the mature lectin (30 kDa a-chain). Since this process is not quantitative, the 16- and 12-kDa fragments (b- and g-chains, respectively) are most probably unligated peptides rather than proteolytic products of the 30-kDa polypeptide. Therefore, the difference in the electrophoretic profiles between native ConBr and the recombinant 34.5-kDa polypeptide produced in P. pastoris, as revealed by Western blotting, is attributed to the absence of further proteolytic processing of pro-ConBr.

In conclusion, when expressed in P. pastoris, prepro-ConBr undergoes a series of transformations, including hyperglycosylation and secretion into the medium. Processing of a fraction of these polypeptides produces a molecular species that has a molecular mass similar to pro-ConBr. No further processing of this precursor was detected. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing that occurs during the biosynthesis of ConBr.

ACKNOWLEDGMENTS

Research supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), and Banco do Nordeste do Brasil (BNB).

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