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Articles by Kadler, K. After three rinses with phosphate-buffered saline Life Technologies, Inc. Latent denotes the latent form of BMP The polyclonal antibody was raised, by Sigma, in rabbits using conventional procedures. Previous Section Next Section.
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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. This is consistent with processing of latent BMP-1 by COS-7 cells occurring after, or during, secretion from the cells.
The C63G and C65A mutants migrated exclusively as the slower migrating reduced 2637. In all the known substrates of BMP-1 the scissile bond resides between a small side-chained residue and an aspartic dw.
Surprisingly, however, the Cys 65 mutant was secreted efficiently. Figure 3 Cleavage of type I procollagen by recombinant BMP-1myc analyzed under non-reducing conditions. We wanted to know whether BMP-1myc cleaved procollagen at the physiological site. This approach is made possible by the fact that the metalloproteinase domain of BMP-1 shares high sequence homology with astacin, whose x-ray crystal structure is known Residue numbers are labeled with an asteriskin blocks of 10 residues and specific for BMP-1 and astacin.
The sequence alignment of astacin and BMP Cys 63 and Cys 66had been mutated were also well secreted. The high degree of 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 Journal of Lipid Research.
Bold indicates homologous substitutions. The amino acid sequence within the N-terminal 10 residues of mature BMP-1 that is recognized by the antibody has not been characterized.
In astacin the active site zinc is pentacoordinated by three histidines, a unique tyrosine residue in the Met turnand a water molecule. We noted that BMP-1 has a lysine residue at position 87 that occurs in all tolloids but is a tyrosine residue in astacin Tyr BMP-1myc was examined by Western blot analysis in which the primary antibody was either the mouse monoclonal anti-c-myc peptide antibody, 9E10, or the rabbit neoepitope polyclonal antibody.
Source of Materials Polymerase chain reaction products were purified with a Qiaquick kit Qiagen. The procollagen content was determined by the sensitive hydroxyproline assay method of Terlink Lane 114 C-labeled type I procollagen 0.
Related Content Load related web page information. In this study we have used site-directed mutagenesis to identify residues in the metalloproteinase domain of BMP-1 that are important for its PCP activity. The pcDNA3 vector containing the cDNA for BMP-1myc was transfected into COS-7 cells, 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 Figure 1 The sequence alignment of astacin and BMP Undigested procollagen P ; containing disulfide-bonded chain migrates near the top of the SDS-gel.
A typical result is shown in Fig. Noteworthy, when astacin binds its substrate, the residues in the substrate N-terminal to the scissile-bound i.
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Bone morphogenetic protein BMP -1, which belongs to the tolloid subgroup of astacin-like zinc metalloproteinases, cleaves the C-propeptides of procollagen at the physiologic site and is, therefore, a procollagen Rs PCP. Cleavage occurs between a specific alanine or glycine residue depending on the procollagen chain and an invariant aspartic acid residue in each of the three chains of procollagen.
This raised the possibility that Cys 85 could form a disulfide bond with a cysteine other than Cys Sequence Analysis In preliminary studies we performed a multiple sequence alignment of 31 members of the astacin family of metalloproteinases using MultAlin 25 data not shown. Furthermore, if these cells synthesized 226237 BMP-1, it was undetectable in assays of procollagen C-proteinase and in Western blotting analyses vs a neoepitope antibody that recognizes the N terminus of mature BMP