Molecular cloning, characterization and gene expression of the full length cDNA encoding the porcine CD11b(αM) and chromosomal localization of the porcine CD11a(αL)-CD11b(αM)-CD11b(αD) gene cluster.

CD11b (α(M)) is a cell-surface glycoprotein mainly expressed on myeloid cells, required for important interactions during the immune response and involved in the pathogenesis of several diseases. The full length cDNA encoding porcine CD11b protein has been cloned and sequenced. Pig CD11b cDNA sequence contains an ORF of 3459 nucleotides that encodes a deduced polypeptide of 1152 amino acid residues that share with CD11b from other species: High % amino acid identity, common domains (α-I, Ca(++) binding, MIDAS), N-glycosylation sites, and the seven FG-GAP tandem repeats. Real time quantitative PCR expression analysis revealed that CD11b transcripts were highly expressed in neutrophils, showing a lower expression in spleen. The CD11a-CD11b-CD11c gene cluster locates on the porcine chromosome region SSC3p15-17.

Two cDNAs coding for the porcine CD51 (αv) integrin subunit: cloning, expression analysis, adhesion assays and chromosomal localization.

CD51 (α(v)) is an integrin chain that associates with multiple ß integrin chains to form different receptor complexes that mediate important human processes. Pigs show substantial physiological, immunological and anatomical similarities to humans, and are therefore a good model system to study immunological and pathological processes. Here we report the cloning and characterization of two cDNAs produced by alternative splicing that encode two different porcine CD51 proteins that differ in five amino acid residues. Pig CD51 cDNAs encode polypeptides of 1046 or 1041 amino acid residues, respectively, that share with other mammalian homologous proteins a high percentage amino acid identity and the functional domains. Expression analysis of CD51 was carried out at two different levels. RT-PCR analysis revealed that both CD51 transcripts were expressed ubiquitously but heterogeneously, with the exception of some platelets in which only the smallest CD51 transcript was detected. A specific monoclonal antibody against a pig CD51 recombinant protein was made and used in the immunohistochemical localization of CD51 proteins. It showed that CD51 was mainly expressed in hematopoietic cells of myeloid linage, epithelial and endothelial cells, osteoclasts, nervous fibers and smooth muscle. Adhesion assays showed that in the presence of Mn(++) pig α(v)-CHO-B2 transfected cells increased their attachment to fibronectin and vitonectin, but not to fibrinogen. Finally, we localized the CD51 gene on the porcine chromosome 15 (SSC15), q23-q26.

Immunohistochemical distribution of the tetraspanin CD9 in normal porcine tissues.

The tetra-membrane-spanning protein, CD9 is a 24-27 kDa cell surface glycoprotein expressed in a wide variety of human cells being involved in a variety of cell processes, including signaling, adhesion, motility, fertilization and tumor cells metastasis. By means of a polyclonal antibody (N1) raised against recombinant swine CD9 protein, we studied the immunohistochemical expression of CD9 on different normal swine tissues. Immunochemistry shows that swine CD9 was distribute in a similar form than in human tissues, being present on epithelial cells of lung, liver, kidney, skin, tonsil, testis (epididymo), gut mucosa, uterus and mama. Furthermore, polyclonal antibody against swine CD9 reacts with white matter from cerebrum and cerebellum, peripheral nerves fibers and Hassal corpuscle from thymus and ovum. Platelets react strongly with our antibody, but monocytes and neutrophils react lightly. These results suggest that CD9 antigen should play a similar functional role in swine and human and therefore studies on CD9 on swine as an animal model would allow new knowledge about its role in adhesion, fertilization and tumor metastasis among other important biomedical processes.

Molecular cloning, expression analysis and chromosome localization of the Tpt1 gene coding for the pig translationally controlled tumor protein (TCTP).

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5'-UTR containing a 5'-terminal oligopyrimidine tract (5'-TOP), and a 3'-UTR with a high CG-content and one AU rich element (ARE). Both 5'-UTR and 3'-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.

Molecular characterization and expression analysis of the gene coding for the porcine beta(3) integrin subunit (CD61).

Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides insight into the physiological function of the porcine beta(3) integrins and should be of importance in understanding the role of this integrin family in biological processes.

Molecular cloning, chromosomal location, and expression analysis of porcine CD14.

CD14 is a membrane-associated glycosylphosphatidylinositol (GPI)-anchored protein that binds lipopolysaccharide (LPS) of Gram-negative bacteria and enables LPS-dependent responses in a variety of cells. In this study a cDNA containing the porcine CD14 coding sequence has been cloned and its complete sequence determined. The amino acid sequence deduced from pig CD14 cDNA encodes a 373 amino acid polypeptide that exhibits 75%, 72%, 69%, 66%, 57% and 56% similarity to CD14 from cow, horse, human, rabbit, mouse and rat, respectively. Structural analysis showed that the porcine CD14 is a membrane glycoprotein with a GPI-anchor site and an extracellular domain containing 11 leucine-rich repeats. In addition, the LPS-binding regions identified in the human CD14 are highly conserved in the N-terminal domain of the porcine sequence. Fluorescence in situ hybridization was used to locate the CD14 gene on the pig chromosome 2, band q28. Expression analysis revealed that porcine CD14 transcripts were detected in all tissues and cells examined, suggesting that the expression of porcine CD14 gene is not restricted to myeloid cell lineage. Finally, we report that LPS stimulation significantly up-regulated CD14 gene expression in porcine alveolar macrophages.

Localization of porcine CD29 transcripts and protein in pig cells and tissues by RT-PCR and immunohistochemistry.

Integrins are heterodimeric cell adhesion proteins with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(1) integrin subfamily are composed of a beta(1) subunit (CD29) non-covalently associated with different alpha subunits to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. Here, we have studied the CD29 expression in porcine tissues and cells at two different levels: expression of the CD29 mRNA by RT-PCR and localization of the protein by immunohistochemistry. CD29 transcripts were detected in a variety of tissues and cells: platelets, PBMC, granulocytes, alveolar macrophages, smooth muscle, intestine, lung, liver, spleen, lymph node, skin, testis, heart, kidney and bone marrow. Our results suggest that CD29 gene transcription occurs in all organs examined, although with different intensities. The precise localization of CD29 protein in paraffin-embedded tissues was detected by using a specific polyclonal antibody indicating that its expression is limited to smooth muscle, epithelium cells, endothelium of blood vessels and myeloid cells and is no detectable in cells of the lymphoid lineage. The distribution of the CD29 in normal tissues provide insight into the physiological function of the porcine beta(1) integrins and should be of importance in understanding the role of this integrin family in pathological processes.