Cloning And Functional Characterization Of A Novel C1r-like Serine Protease, CLSP

Dendritic cells (DC) are the most potent professional antigen-presenting cells (APC), required for the initiation of immune response. For all that the mechanism of DC functions are not fully elucidated. Our studies are aiming at obtaining information about important molecules involved in DC functions by focusing on the cloning of novel genes expressed in DC so as to understand molecular mechanisms underlying the unique capacity of DC.The complement system, a key and conserved system of innate immunity, provides a rapid and efficient means of deleting invading microorganisms. Almost one-third of all proteases can be classified as serine proteases, including complement subcomponent Clr/Cls, mannose-associated serine proteases (MASPs), ovochymase, spermadhesin, type II transmembrane serine proteases (TTSPs) etc. These proteins are involved in diverse biological processes, including developmental processes such as complement activation, ovulation, fertilization, tissue remodeling, cellular migration, cancer invasion and metastasis, intestinal digestion, embryogenesis, or organogenesis. Clr is a complement serine protease, composed of two CUB (Clr/Cls, an embryonic sea urchin protein Uegf, and Bone morphogenetic protein I) domains, one EGF domain and one serine protease domain. Usually, Clr is binding to C1INH (Cl inhibitor). When immune complex (IC) binds to Clq, C1INH is detached form Clr, and then the serine protease is activated, and then the complement classical pathway is activated.We detailed here a novel gene CLSP, isolated from the DC cDNA library by large-scale random sequencing. Full length of CLSP contains 3345 bp, with an ORFof 1464 bp. The full ORP encodes a 487-amino acid protein with a calculated molecule weight of 53.484 kDa and an isoelectric point of 6.75. At the primary sequence level, it shared high homology with Clr, so was named Clr-like serine protease, CLSP. The CLSP sequence has been submitted to GenBank/EMBL (GenBank Accession Number AF178985).The CLSP protein shared roughly 25-40% identity and 40-55% similarity to complement subcomponent Clr, Cls, mannose-associated serine protease-1 (MASP-1), MASP-2, and MASP-3. Hydrophobicity analysis predicted a 36-residue hydrophobic signal peptide for secretion in the N-terminus, and no transmembrane region was present, suggesting it might be a type of secretory protein. Some generic and atipical N-glycosylation sites were present in CLSP, suggesting that the protein might represent a glycoprotein. The CLSP protein contained one CUB (Clr/Cls, an embryonic sea urchin protein Uegf, and Bone morphogenetic protein I) domain from 39th to 164th ammo acids, which is known to be involved in protein-protein or protein-substrate interaction, and a trypsin-like serine protease domain positioned from 244th to 483rd amino acids. The CUB domain and serine protease domain 01 CLSP closely resembled the corresponding domains of Clr (48% and 58% identity, respectively). The conserved cystein residues also present in the CUB domain of CLSP at position of 36, 94 and 112. For trypsin-like serine protease domain, the presence of His (H) -Asp (D) -Ser (S) representing a serine protease catalytic triad were at position of 283, 339, 436, respectively. Moreover, Asp immediately preceding the active site Ser is necessary for proper folding of the catalytic site. The human CLSP cDNA corresponded to Unigene cluster HS.415792 located on human chromosome 12pl3.31. Nucleotide sequence alignment of CLSP and this sequence enabled us to demonstrate the genomic structure of the CLSP gene with appropriate exon-intron boundaries, which contained 6 exons and 5 introns. The CLSP gene is located approximately 3 kb and 90 kb centromeric to the Clr and Cls gene, respectively, and their genes form a short cluster in a region of about 115kb in 12pl3.The cluster gene arrangement and the sequence similarities of the CUB and trypsin-like serine protease domain of CLSP with other complement component 1 subcomponents supported a common evolutionary history by consecutive gene dup...