title: Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene.
abstract: OBJECTIVE
  Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5).
  
  
  METHODS
  The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry.
  
  
  RESULTS
  As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT-PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes.
  
  
  CONCLUSIONS
  The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.
authors.affiliation: Department of Animal Science, North Carolina State University, Raleigh, NC, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Sommer
authors.forename: Venkata R M
authors.initials: VR
authors.lastname: Chavali
authors.forename: Sean G
authors.initials: SG
authors.lastname: Simpson
authors.forename: Radha
authors.initials: R
authors.lastname: Ayyagari
authors.forename: Robert M
authors.initials: RM
authors.lastname: Petters
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D015922:Complement C1q / Q000235:genetics*; D017242:Complement Factor H / Q000378:metabolism; D005123:Eye / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D034421:Sus scrofa / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D008565:Membrane Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D015922:Complement C1q; D017242:Complement Factor H