cDNA Cloning and Overexpression of Cell Surface Subtilisin‐Like Proteases (SSP) of Pneumocystis carinii

P. carinii are coated by abundant surface proteins called MSG for major surface glycoproteins which play vital roles in pathobiology. MSGs are encoded by a family of polymorphic genes, which often cluster with a protein coding sequence named OW3 [3]. In this study, we cloned ORF3-cDNAs and discovered that they encode subtilisin-like proteases with polymorphic protein sequences. The deduced amino acid sequences were 50% identical compared with the catalytic domain of eukaryotic subtilisin-like proteases (e.g., yeast Kex2 and human furin), and contained a C-terminal anchoring sequence in the cell membrane (hence referred to as SSP for Surface Subtilisin-like Protease), showing a unique features of surface polymorphic proteases associated with the MSGs of P. carinii. MATERlALs AND METHODS. A P. carinii cDNA library [2] was screened by plaque hybridization using an ORF3 probe cloned i n MW111 [3]. The positive clones were subcloned into plasmid pUC118 or pUC119, and sequenced. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession number AB003497-AB003500. Two-thirds of the O W 3 cDNA (nucleotide positions 4161579) encoding the catalytic domain of subtilisinlike protease was cloned into expression plasmid pET29b (Novagen) and the protein was tagged at its C-terminus with a hexa-histidine sequence. E. coli strain HMS174 @E3) transformed with this construct synthesized a His6-SSP upon addition of IPTG (0.4 mM) by T7 RNA polymerase driven expression. The induced lysate was subjected to SDS-polyacrylamide gel electrophoresis. and detected by Coomassie staining or by standard Western blot techniques using an Ni-NTA-alkaline phosphatase conjugate (Qiagen). RESULTS AND DISCUSSION. The nucleotide sequence analyses of ten ORF3-cDNA clones isolated in this study revealed that they are similar but not identical, showing polymorphism in the overlapped sequences. This is consistent with the previous finding that the OW3 DNA probe hybridized to multiple restriction fragments i n Southern blot hybridization [3]. The cDNA analysis indicated that OW3 has extended 5'-coding regions containing introns. The deduced full-length product of ORF3cDNA is composed of 790 amino acids. By database search, O W 3 showed high protein sequence similarity to subtilisin-like proteases of eukaryotes: 51 and 47% identity t o the catalytic domain of Saccharomyces cerevisiae Kex2 and human furin, respectively. Subtilisin-like proteases are known to cleave polypeptides at pairs of basic amino acid residues, and conserve sequences around the active site residues of Asp, His and Ser. The OW3 protein has a conservation pattern, showing that the cloned cDNA encodes P. carinii subtilisin-like protease (Fig. 1). In addition to sequence homology, P. carinii subtilisin-like protease shares other properties common to the eukaryotic subtilisin-like protease family: it has a putative signal sequence at its N-terminus as well as a possible autocatalytic site, lysyl-arginine, at the upstream of the catalytic domain. As shown previously [3], P. carinii subtilisin-like protease has a proline-rich region and a serinehhreonine-rich re*