Molecular and Biochemical Parasitology, 43 (1990) 289-292 289

Elsevier

MOLBIO 01447

Short Communica t ion

Cloning and expression of Schistosoma mansoni protein Sm32 in a baculovirus vector

Richard Felleisen I , Ewald Beck 1, Magda Usmany 2, Just Vlak 2 and Mo-Quen Klinkert 3

I Zentrum fiir Molekulare Biologie, University of Heidelberg, Heidelberg, F.R.G., and 2Department of Virology, Agricultural University, Wageningen, Wageningen, The Netherlands

(Received 27 June 1990; accepted 17 August 1990)

Key words: Schistosoma mansoni; Expression; Baculovirus; Glycosylation

The entire coding sequence of a Schistosoma mansoni protein termed Sm32 [1] was engineered into the genome of Autographa californica nu- clear polyhedrosis virus (AcNPV). Sm32 has been studied in several laboratories as a candidate anti- gen for immunodiagnosis of schistosomiasis [2-4]. Moreover, it has been proposed to be a digestive protease, playing a role in haemoglobin degrada- tion [5]. The non-fused schistosome antigen was expressed in insect cells under the control of the strong polyhedrin promoter. We were interested in investigating the suitability of insect cells as an expression system for efficient production of pro- teins from cloned schistosome genes. The insect cell-synthesised product was analysed for modifi- cations through glycosylation and proteolytic pro- cessing, in comparison with the native antigen and the in vitro translated Sm32.

The full-length cDNA insert M2-16 from )~gtl0 for Sm32 [6] was subcloned in an RNA expres- sion vector pSP65/NcoI [7] (Fig. 1A). pSP-Sm32 was derived by deleting the non-coding region of Sm32 at the N-terminus for more efficient ex- pression of the RNA in rabbit reticulocyte trans- lation systems (Fig. 1B). The resulting fragment was cloned into the plasmid transfer vector pBC3,

Correspondence address: M.-Q. Klinkert, ZMBH, University of Heidelberg, Im Neuenheimer Feld 282, 69 Heidelberg, F.R.G.

derived from pAcJR2 [8] (Fig. 1C). Transfer of Sm32 cDNA by the chimaeric transfer vector pBC-Sm32 to the AcNPV genome was achieved by co-transfection of Spodoptera frugiperda cells using the calcium phosphate precipitation tech- nique [9]. A recombinant virus using standard methods [9] was isolated and referred to as BC- Sm32.

AcNPV recombinant viruses were produced in S. frugiperda cells using TNM-FH medium [10]. Insect cells (at a cell density of 5 × 105 ml - t ) were infected for 4 days with BC-Sm32. Infected cells were seeded in a volume of 30 ml in 850-ml flasks (NUNC) and incubated at 28°C. Follow- ing harvest, total cell proteins were analysed on SDS-polyacrylamide gels. The yield of recombi- nant protein was estimated to be approximately 1% of total cell proteins. The reactivity was anal- ysed by Western blotting using rabbit antiserum specific for Sm32 (Fig. 2A). The production of this antiserum has been described elsewhere [11].

No reaction with Sm32-specific antibodies was detected in the lysates of uninfected cells (lane 1), nor in their culture supernatants (lane 2). How- ever, using lysates from BC-Sm32 infected S. frugiperda cells, three proteins with approximate sizes of 40, 41.5 and 43 kDa were recognised (lane 3). Media from BC-Sm32-infected cell cul- tures had no detectable Sm32 antigen, indicating

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A Pstl Ncol

SP6 Promoter

Drag N¢ol

pSP-M2-16

B SP6 Promoter

PstI Ncol

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C PstI Neol

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pBc-SmSZ

Fig. 1. Construction of chimaeric transfer vector pBC-Sm32. (A) The Ncol cDNA fragment M2-16 from Agtl0, which contains the entire coding sequence of Sm32, was subcloned in pSP65/NcoI to yield pSP-M2-16. (B) Artificial ATGs contained in the adaptor sequence tAD) of pSP-M2-16 and the non-coding region were eliminated in a second step using the Ncol site on the linker of pSP65/NcoI and the DraI site on the Sm32 fragment. Deletion of the Ncot/DraI Sm32 fiagment resulted in the construct pSP-Sm32. (C) The AcNPV transfer vector pBC3 was used as an acceptor for the 1.5-kb PstI and Ncol fragment of pSP-Sm32, pBC3 contains the polyhedrin promoter and the first 33 nucleotides of the coding sequence of the polyhedrin structural gene (PCS), which read: ACGCCGGATTATTCATACCGTCCCACCATCGGG. The polyhedrin AUG translation initiation codon was converted to ACG by site directed mutagenesis, and so translation of the mRNA derived from the polyhedrin promoter initiates at the schistosome AUG

to yield non-fused schistosome protein Sm32. The resulting chimaeric transfer vector was named pBC-Sm32.

that the protein was not secreted (lane 4). Cells infected with wild-type AcNPV also did not react with Sm32-specific antibodies (lane 5).

The nucleotide sequence of Sm32 predicts an open reading frame comprising 428 amino acid residues and could give rise to a protein of 46 kDa. However, the mature protein has an apparent size of 32 kDa. By sequencing of the carboxy-terminus of the purified mature protein with carboxypepti- dase Y, it was shown that 138 C-terminal amino acids predicted from the ORF are lacking [12]. Consequently, it can be argued that Sm32 is ini- tially synthesised as a large precursor, which after undergoing co-translational modifcations during its biosynthesis, is processed into the mature form. It therefore seemed reasonable to assume that the three polypeptides of 40, 41.5 and 43 kDa ex- pressed by the baculovirus vector system repre- sent differentially glycosylated and/or processed intermediates of the precursor protein.

Two hypothetical N-glycosylation sites are present in the primary sequence of Sm32 [1]. Glycosylation of Sm32 in vivo was demonstrated following treatment of schistosome extracts with

endoglycosidase H [13] and analysis by Western blotting in the presence of anti-Sm32 antiserum (Fig. 2B). In the untreated extracts anti-Sm32 an- tiserum reacted with a 32-kDa protein (lane 1). In contrast, after endoglycosidase H treatment, two proteins estimated to be approximately 30 and 28 kDa were identified, the former interpreted as a protein lacking one carbohydrate moiety and the latter representing the fully deglycosylated form (lane 2). No precursor molecule was detected in schistosome extracts, as might be expected from the size of the open reading frame.

In support of the above results, in vitro glyco- sylation of this protein at two sites has also been asserted. The major translation product of 46 kDa, derived from the RNA synthesised from clone pSP-Sm32 was converted to a 50-kDa compo- nent in the presence of canine microsomal mem- branes [14], while treatment with endoglycosidase H yielded a shift back to its original position of 46 kDa (data not shown).

The products derived from insect cells were also examined for the presence of carbohydrate side chains (Fig. 2B). In the untreated extract, anti-

1 2 3 4 5 M i i i i i i i 1 2 3 4 M

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~ i ~ - - 75 --66,2

- - 3 2

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291

--42,7

--31,0

- - 1 7

--21,5

--14,4

. . . . . . . . . . . . A B

Fig. 2. (A) Western blot analysis of total cell extract of infected and uninfected S.frugiperda cells. Proteins were resolved in 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose filter and probed with rabbit anti-Sin32 antibodies. (1) Uninfected cells (2) cul- ture supernatant from uninfected cells, (3) recombinant Sm32 from BC-Sm32-infected cells, (4) media from BC-Sm32 infected cells, and (5) AcNPV infected cells were analysed. (B) Western blot analysis of (I) untreated schistosome extract, (2) endoglycosidase H-treated schistosome extract, (3) baculovirus-synthesised products before treatment with endoglycosidase H and (4) after treatment.

Sm32 antibodies reacted with the protein triplet (lane 3). Endoglycosidase H-treated extract probed with the serum revealed the presence of the 40- kDa protein only (lane 4). This protein most likely corresponds to a fully deglycosylated form, while the 41.5- and 43-kDa products probably repre- sent proteins containing one and two carbohydrate moieties, respectively. Thus, we have shown the presence of differentially glycosylated forms of Sm32 in insect cells.

The question remaining open is why the Sm32 produced in insect cells is still considerably larger than the native molecule. Preliminary results sug- gest that proteolytic cleavage of the baculovirus- synthesised products and the in vitro transla- tion product of Sm32 does not occur, even af- ter prolonged incubation. Assuming that part of the cleavage mechanism is cell type- and species- dependent, then the absence of S. mansoni-specific factor(s) in these systems for complete process- ing of the polypeptide could explain the exis-

tence of forms with different molecular weights. Interestingly, two reports have described a 41-kDa species occasionally co-purifying with the native 32-kDa protein [15,16], the former being consis- tent in size with the proteins produced in insect cells. We therefore speculate that processing of Sm32 is a multi-step procedure, involving a gly- cosylated precursor molecule, which can be par- tially cleaved under physiological conditions to an intermediate of approximately 40 kDa, ma- ture Sm32 being released in a further processing step.

Recent evidence has suggested that Sm32 may be a digestive enzyme, possibly participating in the degradation of haemoglobin [5]. Given that most proteases are first made as inactive proen- zymes, it is not surprising that larger forms of Sm32 exist. Future efforts will be directed towards elucidating the factors responsible for the partic- ular processing steps of Sm32, based on our un- derstanding of its expression and regulation using

292

insect cells/baculoviruses and in vitro translation as model systems.

References

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