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Rabbit liver microsomal endopeptidase with substrate specificity for processing proproteins is structurally related
S Kawabata1, K Nakagawa, T Muta
1Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Abstract:
The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E. W. (1992) J. Biol. Chem. 267, 10331-10336). These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones. A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries. The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis. MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases. Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence. Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar. MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity).
Insights
Rabbit liver microsomes contain a novel endopeptidase (MEP) that cleaves specific Arg-containing peptides. The study isolated and characterized the cDNA for MEP, revealing its structure and homology to other metallopeptidases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Rabbit liver microsomes contain an endopeptidase (MEP) with specific substrate requirements.
- MEP cleaves peptides with Arginine residues at P1 and P4 positions, relevant for proprotein processing.
- This enzyme's activity is crucial for understanding the maturation of various proproteins.
Purpose of the Study:
- To isolate and characterize the cDNA encoding rabbit liver microsomal endopeptidase (MEP).
- To determine the protein sequence, active site, and structural features of MEP.
- To investigate the homology of MEP with other known peptidases.
Main Methods:
- Isolation of overlapping cDNA clones from rabbit liver lambda gt10 cDNA libraries.
- Sequencing of the cDNA and determination of the open reading frame.
- Amino acid sequence analysis for protein confirmation, active site identification, and structural prediction.
- Hydropathy plotting and N-glycosylation site analysis.
Main Results:
- A 3507-base pair cDNA was obtained, coding for a 704-amino acid protein, with 406 residues confirmed.
- MEP possesses a putative zinc metallopeptidase active site (-His-Glu-X-X-His-).
- MEP is hydrophilic, lacks a transmembrane domain and signal sequence, and is not N-glycosylated.
- Significant homology was found with rat testes metalloendopeptidase 24.15 (60% identity) and other peptidases.
Conclusions:
- The cDNA for rabbit liver microsomal endopeptidase (MEP) has been successfully isolated and sequenced.
- MEP is a novel mammalian zinc metallopeptidase with a distinct substrate specificity.
- MEP shares homology with other metallopeptidases, suggesting conserved functions across species.