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Molecular Characterization of a Saposin-Like Protein Family Member Isolated from Bovine Lymphocytes

¹ Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing 48824
² Department of Veterinary Science, Penn State University, University Park 16802

Corresponding author: Lorraine M. Sordillo; e-mail: sordillo{at}msu.edu.

Human and porcine T lymphocytes and natural killer (NK) cells produce antibacterial proteins that belong to the saposin-like family of proteins (SAPLIP). The objective of this study was to determine if a bovine homolog of SAPLIP exists in lymphocytes that exhibit antibacterial activity. Following stimulation with IL-2, bactericidal activity against Staphylococcus aureus was detected to some extent in most major subpopulations of T lymphocytes including CD4⁺, CD8⁺, CD3⁺, and WC1⁺ T lymphocytes. However, the majority of antibacterial activity was observed in the CD2⁺CD3^– lymphocytes, which are similar phenotypically to NK cells. A partial sequence of a bovine SAPLIP was generated using low specificity primers designed from regions of homology between other SAPLIP including porcine NK-lysin and human granulysin. Enhanced expression of the bovine lysin gene was detected in mRNA isolated from IL-2–stimulated CD2⁺CD3^– lymphocytes. The partial cDNA sequence was then used to make gene specific primers for a rapid amplification of cDNA ends (RACE) procedure that provided repeatable 5' and 3' cDNA ends. By examining overlapping regions from the RACE procedure, full-length sequence information was obtained for the bovine lysin homologue. Conceptual translation of the cDNA demonstrated conserved similarities to known SAPLIP members. Further characterization of the bovine lysin may facilitate its use in protecting dairy cattle against bacterial infections.

Key Words: saposin-like • lymphocyte • bovine

Abbreviation key: CF = cell free, GSP = gene-specific primers, HBSS = Hank’s balanced salt solution, NK = natural killer, ORF = open reading frame, PBMC = peripheral blood mononuclear cells, QC RT-PCR = quantitative competitive reverse transcription PCR, RACE = rapid amplification of cDNA ends, SAPLIP = saposin-like protein, TIGR = The Institute for Genomic Research