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Molecular and biological characterization of rabbit mannan-binding protein
1Department of Food Microbiology, Osaka Prefectural Institute of Public Health, Higashinari, Japan.
Abstract:
Mannan-binding protein (MBP) is a member of the collectin family of protein. There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP. It was considered that the loss of one MBP gene occurred during hominoid evolution. In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically. Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical. A serum MBP has a higher ability to activate complement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does. cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here. Northern blot analysis showed that mRNA transcripts of about 0. 9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.
Insights
Researchers characterized rabbit mannan-binding protein (MBP), finding liver and serum forms share identical sequences but differ in complement activation and viral inhibition. Gene loss occurred beyond hominoid evolution.
Area of Science:
- Immunology
- Molecular Biology
- Evolutionary Biology
Background:
- Mannan-binding protein (MBP) is a collectin family protein with two types (MBP-A, MBP-C) in many mammals, but only one in humans and chimpanzees.
- Previous studies suggested a loss of one MBP gene during hominoid evolution.
- This study focuses on characterizing rabbit MBP, investigating potential differences between liver and serum forms.
Purpose of the Study:
- To biologically and genetically characterize two types of rabbit MBP: liver and serum MBP.
- To compare their molecular weight, amino acid sequences, complement activation abilities, and hemagglutination inhibition.
- To elucidate the evolutionary history of MBP gene loss.
Main Methods:
- SDS-PAGE under reduced conditions to determine molecular weight.
- Amino acid sequencing of liver and serum MBP.
- Complement activation and hemagglutination inhibition assays.
- cDNA cloning from a rabbit liver library using mouse MBP-C as a probe.
- Northern blot analysis to detect mRNA transcripts.
- Phylogenetic tree analysis of MBP and other collectins.
Main Results:
- Rabbit liver and serum MBP are identical 32 kDa proteins.
- Serum MBP shows higher complement activation, while both inhibit influenza virus hemagglutination equally.
- cDNA analysis confirmed the protein sequence and identified a 22-residue signal peptide.
- MBP mRNA transcripts (0.9 and 3.0 kb) are exclusively expressed in the liver.
- Phylogenetic analysis suggests MBP gene loss occurred after bird-mammal divergence, not solely in hominoids.
Conclusions:
- Rabbit possesses a single MBP gene, with liver and serum forms arising from differential expression or post-translational modification.
- The functional differences highlight tissue-specific roles of MBP.
- MBP gene loss is a more ancient evolutionary event than previously thought, predating hominoid evolution.