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Published on: September 22, 2020
Molecular cloning, chromosomal location, and expression analysis of porcine CD14
Gema Sanz1, Eva Pérez, Angeles Jiménez-Marín
1Unidad de Marcadores Genéticos Moleculares, Departamento de Genética, Universidad de Córdoba, Campus de Rabanales, 14071 Córdoba, Spain.
Insights
Researchers cloned and sequenced porcine CD14, a protein crucial for lipopolysaccharide (LPS) response. The study details its structure, gene location, and expression patterns, finding it widely distributed and upregulated by LPS in macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- CD14 is a key membrane protein involved in lipopolysaccharide (LPS) recognition and response in various cells.
- Understanding CD14's role in different species is vital for comparative immunology and disease research.
Purpose of the Study:
- To clone and sequence the porcine CD14 gene.
- To analyze the structural and functional characteristics of porcine CD14.
- To investigate the gene's chromosomal location and expression patterns in pigs.
Main Methods:
- CDNA cloning and sequencing of porcine CD14.
- Amino acid sequence comparison with other species.
- Structural analysis of protein domains, including leucine-rich repeats and GPI-anchor site.
- Fluorescence in situ hybridization (FISH) for gene localization.
- Gene expression analysis in various tissues and cell types, including LPS stimulation of alveolar macrophages.
Main Results:
- The porcine CD14 cDNA sequence was determined, encoding a 373-amino acid protein with significant similarity to CD14 from other mammals.
- Structural analysis revealed conserved LPS-binding regions and a GPI-anchor site.
- The CD14 gene was mapped to pig chromosome 2, band q28.
- Porcine CD14 transcripts were detected in all examined tissues, indicating broad expression beyond myeloid cells.
- LPS stimulation significantly increased CD14 gene expression in porcine alveolar macrophages.
Conclusions:
- The study provides a comprehensive characterization of porcine CD14, including its sequence, structure, and genomic location.
- Porcine CD14 exhibits conserved functional domains and a wide tissue distribution.
- The findings contribute to understanding innate immunity mechanisms in pigs and offer insights into CD14's evolutionary conservation.
Abstract:
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.
