Lee, N A

Abstract The existence of a murine homologue of the major basic protein (MBP) found in human eosinophil granules was initially hypothesized from structural similarities at the electron microscopic level. The results presented in this study have extended these observations by describing the identification/purification of a mouse MBP (mMBP) and the cloning of the gene encoding this eosinophil granule protein. Using protein purification methodologies with extravascular eosinophils, an mMBP homologue has been identified on the basis of strong (64%) N-terminal sequence homology with the mature human MBP (hMBP). Since hMBP results from a proteolytic cleavage of a precursor molecule, this sequence conservation suggests that the mouse granule protein is processed by a similar mechanism. The gene encoding mMBP was isolated using a hMBP cDNA clone as a heterologous probe in low criteria screens of mouse genomic and cDNA libraries. The genomic structure and nucleotide sequence of the mMBP exons are well conserved with the human gene, although homology alignments of the encoded proteins show that extensive sequence conservation occurs only in the mature portion of the MBP molecules. Expression data demonstrate that this gene is transcriptionally active in tissues containing eosinophil progenitor cells, such as femoral bone marrow. Genomic Southern blots using the mMBP gene at reduced stringency reveal the potential existence of a second, more divergent MBP-like sequence in the mouse. This suggests that, as with guinea pigs, the mouse genome may also encode the eosinophil major basic protein from more than one gene.

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).