Abstract
Alpha-2-macroglobulin (A2M) binds proteases, thereby acting as defense barriers against pathogens in the plasma and tissues of vertebrates and invertebrates. Quantitative real-time polymerase chain reaction (PCR) and the isobaric tags for relative and absolute quantitation method were used to determine the expression levels of A2M mRNA and proteins in mastitis-infected mammary tissues. A2M mRNA and protein expression were significantly higher in mastitis-infected mammary tissues than those in healthy tissues. We also identified 23 novel A2M splice variants in the bovine mammary tissues using reverse transcription PCR combined with clone sequencing. These splice variants predominantly affected the bait region, the inhibitory region, and the thioester region of the protein, which have the functional key roles in inhibiting the proteases of pathogens. Genomic sequencing analysis revealed a nonsynonymous c.3535A>T single-nucleotide polymorphism (SNP) in exon 29, which is located within a putative exonic splice enhancer and may be the reason why the A2M gene produces the aberrant splice variant A2M-AS4. Our findings suggest that the A2M gene can play its role by alternative splicing mechanism and it may be of significance against mastitis. This study provides clues to better understand the function of the bovine A2M gene and the effects of the exonic SNP on the production of aberrant splice variants.
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Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (No. 31000543), the Major Project of National Transgene in China (2011ZX08007-001), the Support Program of the Ministry of Science and Technology, P. R. China (2011BAD19B02 and 2011BAD19B04), the Program of National Cow Industrial Technology System (CARS-37), and the Project of Agricultural Fine Breed from the Department of Science and Technology of Shandong Province (2010LZ10-02).
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Wang, X., Huang, J., Zhao, L. et al. The exon 29 c.3535A>T in the alpha-2-macroglobulin gene causing aberrant splice variants is associated with mastitis in dairy cattle. Immunogenetics 64, 807–816 (2012). https://doi.org/10.1007/s00251-012-0639-8
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DOI: https://doi.org/10.1007/s00251-012-0639-8