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Guinea pig alpha 1-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase
K Yoshida1, Y Suzuki, K Yamamoto
1Department of Biochemistry, Kinki University School of Medicine, Osaka, Japan. kojiy@med.kindai.ac.jp
Abstract:
cDNA encoding alpha 1-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavia porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gln rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change.
Insights
Researchers sequenced guinea pig alpha 1-microglobulin/bikunin (AMBP) cDNA, finding homology to other mammals but a unique C-terminal domain variation. AMBP mRNA was widely distributed, with levels unchanged during inflammation.
Area of Science:
- Molecular Biology
- Mammalian Genetics
- Biochemistry
Background:
- Alpha 1-microglobulin/bikunin (AMBP) is a multifunctional protein with roles in protease inhibition.
- Understanding mammalian AMBP diversity is crucial for its functional characterization.
Purpose of the Study:
- To clone and sequence the cDNA encoding alpha 1-microglobulin/bikunin (AMBP) in guinea pigs (Cavia porcellus).
- To investigate the tissue distribution of AMBP mRNA.
- To analyze AMBP mRNA expression levels during inflammation.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to amplify and sequence AMBP cDNA.
- RT-PCR and nested PCR were employed to detect AMBP mRNA distribution.
- Quantitative RT-PCR was used to compare AMBP mRNA levels in normal and inflamed liver tissue.
Main Results:
- The guinea pig AMBP sequence showed 69-76% amino acid identity to other mammals, featuring two Kunitz-type trypsin inhibitor domains.
- A notable difference was observed in the P1 residue of the C-terminal inhibitor domain (Gln instead of conserved Arg).
- AMBP mRNA was detected in multiple tissues including liver, pancreas, and leukocytes, but not in brain or heart.
- Inflammation did not significantly alter AMBP mRNA levels in guinea pig liver.
Conclusions:
- The study provides the first molecular characterization of guinea pig AMBP, revealing conserved and divergent features compared to other mammals.
- The widespread expression of AMBP mRNA suggests diverse physiological roles beyond the liver.
- AMBP mRNA levels appear stable under inflammatory conditions in guinea pig liver.