Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.
Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.
Aoki
I
I
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Shindoh
Y
Y
Nishida
T
T
Nakai
S
S
Hong
Y M
YM
Mio
M
M
Saito
T
T
Tasaka
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics* / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000502:physiology*; D006168:Guinea Pigs; D006636:Histamine Release; D008297:Male; D008407:Mast Cells / Q000187:drug effects; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012260:Ribonucleases; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D009841:Oligonucleotides; D012260:Ribonucleases; D002118:Calcium