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In this study we used information from the three-dimension structure of astacin, the x-ray crystal structure of astacin in complex with a transition state inhibitor 20and the primary structures of different members of the astacin family to identify residues in BMP-1 that might account for the procollagen C-proteinase activity of BMP Although the metalloproteinase domains of astacin and BMP-1 are homologous and presumably have a similar tertiary structure, it is not obvious why BMP-1 cleaves scissile bonds between a small side chained residue and an aspartic acid.
We showed that substitution of alanine for Glu 94 eliminated the PCP activity of BMP-1 and confirmed that this glutamic acid residue is essential for catalytic activity of this enzyme. A minor change was that laser densitometry of film exposed to dried gels was replaced by image plate quantitation of the 14 C-labeled proteins.
Therefore, understanding the catalytic mechanism of this enzyme is relevant to studies of animal development and tissue organization. Cleavage of type I procollagen by recombinant BMP-1myc analyzed under non-reducing conditions. Section solely to indicate this fact. Residue numbers are labeled with an asteriskin blocks of 10 residues and specific for BMP-1 and astacin. Pwo DNA polymerase was used to minimize base misincorporation during the polymerase chain reactions.
After three rinses with phosphate-buffered saline Life Technologies, Inc.
This is a direct analogy with what happens in astacin when Trp 65 the equivalent of Cys 66 in BMP-1 backs onto the P1 position of the astacin substrate. The x-ray crystal structure of astacin shows a disulfide bond between a cysteine in the upper edge of the active site cleft and a cysteine buried in the body of the metalloproteinase domain Recombinant BMP-1 was assayed for procollagen C-proteinase activity using human 14 C-labeled type I procollagen substrate and analysis of the cleavage products on SDS gels as described 3.
In this study we have used site-directed mutagenesis to identify residues in the metalloproteinase domain 2637 BMP-1 that are important for its PCP activity. In brief, a peptide corresponding to the 10 N-terminal residues of the mature BMP-1 protein after removal of the prodomain was conjugated to keyhole limpet hemocyanin and subsequently used to immunize two separate rabbits.
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The pcDNA3 vector containing the cDNA for BMP-1myc was transfected into COS-7 62237, and the conditioned culture medium and the cell lysate were analyzed by Western blotting using the 9E10 antibody which detects the c-myc tag at the C terminus of the molecule and the neoepitope antibody which was raised to a peptide corresponding to the 10 residues at the N terminus of mature BMP The results showed that BMP-1myc was secreted into the culture medium mostly as the mature enzyme.
Journal of Lipid Research. The preparations were then examined in assays of procollagen C-proteinase.
Undigested procollagen P ; containing disulfide-bonded chain migrates near the top of the SDS-gel. The importance of BMP-1 in tissue assembly is exemplified in the BMP-1 knockout mouse, which dies soon after birth from failure of ventral body wall closure associated with abnormal collagen fibrillogenesis 5.
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Plasmids were extracted with a Qiaprep spin miniprep kit Qiagen. The most likely candidate was Cys 63because based on structure comparison with astacin in complex with an inhibitor, its side chain is oriented toward the Cys 85 residue Also, BMP-1 266237 in which the other two cysteine residues on the active site edge strand, i.
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The levels of BMP-1myc were quantitated by dz densitometry of enhanced chemiluminescence fluorograms exposed to pre-flashed film. The observation that the culture medium of cells transfected with the E94A mutant did not contain detectable PCP activity also validated the use of COS-7 cells as a good model cell system in which to express recombinant BMP Briefly, a DNA fragment was amplified using the Xcm Forward primer and the antisense mutant primer, and an overlapping fragment was amplified using the sense mutant primer and the downstream Blp Reverse primer.
262377 C63G and C65A mutants migrated exclusively as the slower migrating reduced form. Dz metalloproteinase domain of astacin is kidney-shaped and has two domains at the N and C termini that are separated by an active site cleft It was first necessary to establish and validate a system capable of expressing recombinant BMP-1 that exhibited PCP activity. The absence of the Cys 63 -Cys 66 disulfide bond in 1 the Cys 66 mutant, 2 the Cys 63 mutant because Dds 66 has 262237 free thiol group in this mutantand 3 the Cys 65 mutant because Cys 85 has bonded with Cys 63 thus leaving a free thiol group on Cys 66 alters the chemical structure of the S1 site and decreases PCP activity.
The study also showed that the introduction of the c-myc tag at the C terminus of BMP-1 did not prohibit subsequent assays of PCP activity.
In BMP-1, this glutamic acid is at amino acid position BMP-1myc and BMP-1 were detected by 226237 blot analysis using anti-c-myc monoclonal antibody 9E10 and the neoepitope antibodyrespectively. In samples containing wild-type BMP-1 and recombinant BMP-1myc the procollagen was converted to a normal intermediate in the conversion of procollagen to collagen containing the N-propeptides but not the C-propeptides.
The high degree dz sequence homology between the metalloproteinase domains of astacin and BMP-1, and the fact that the metalloproteinase domains are similar in size, suggests that the structure of the metalloproteinase domain of BMP-1 is similar to that of astacin The amino acid sequence within the N-terminal 10 residues of mature BMP-1 that sd recognized by the antibody has not been characterized.
This loop contains Lys This is consistent with processing of latent BMP-1 by COS-7 cells occurring after, or during, secretion from the cells. Cleavage of type I procollagen by recombinant BMP-1myc analyzed under reducing conditions.
The polyclonal antibody was raised, by Sigma, in rabbits using conventional procedures. Cell lysates contained only the latent form of BMP The culture media of the cells were dss by Western blot analysis using the 9E10 monoclonal antibody.