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Biomedical subjects

W V Arnold

Publications and source records attributed to W V Arnold.

7 recordsLinked to original sources
PubMed1998

Recombinant procollagen II: Deletion of D period segments identifies sequences that are required for helix stabilization and generates a temperature-sensitive N-proteinase cleavage site.

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 degreesC as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 degreesC lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 degreesC lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 degreesC that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the alpha1(II) chain.

Binding Sites↗
PubMed1997

A cDNA cassette system for the synthesis of recombinant procollagens. Variants of procollagen II lacking a D-period are secreted as triple-helical monomers.

Currently there is a lack of experimental systems for defining the functional domains of the fibrillar collagens. Here we describe an experimental strategy that employs the polymerase chain reaction (PCR) to create a series of cDNA cassettes coding for seven separate domains of procollagen II. The system was used to prepare novel recombinant procollagens II from which one of the four repetitive D-periods of the triple helix was deleted. Four constructs, each lacking a different D-period, were expressed in stably transfected mammalian cells (HT-1080). Truncated procollagens of the predicted size were recovered from the medium. All were triple-helical as assayed by circular dichroism. Therefore, deletion of a complete D-period containing 234 amino acids does not destabilize the triple helix of homotrimeric collagen II as much as some naturally occurring mutations in the heterotrimeric monomer of collagen I that delete shorter sequences or that convert obligate glycine residues to residues with bulkier side chains. Moreover, the results suggest that the strategy developed here can be used to map in detail the binding sites on fibrillar collagens for other components of the extracellular matrix and for the binding, spreading and signaling of cells.

Animals↗
PubMed1996

The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

Amino Acid Sequence↗