A CDNA CASSETTE SYSTEM FOR THE SYNTHESIS OF RECOMBINANT PROCOLLAGENS - VARIANTS OF PROCOLLAGEN-II LACKING A D-PERIOD ARE SECRETED AS TRIPLE-HELICAL MONOMERS
Wv. Arnold et al., A CDNA CASSETTE SYSTEM FOR THE SYNTHESIS OF RECOMBINANT PROCOLLAGENS - VARIANTS OF PROCOLLAGEN-II LACKING A D-PERIOD ARE SECRETED AS TRIPLE-HELICAL MONOMERS, Matrix biology, 16(3), 1997, pp. 105-116
Currently there is a lack of experimental systems for defining the fun
ctional domains of the fibrillar collagens. Here we describe an experi
mental strategy that employs the polymerase chain reaction (PCR) to cr
eate a series of cDNA cassettes coding for seven separate domains of p
rocollagen II. The system was used to prepare novel recombinant procol
lagens II from which one of the four repetitive D-periods of the tripl
e helix was deleted. Four constructs, each lacking a different D-perio
d, were expressed in stably transfected mammalian cells (HT-1080). Tru
ncated procollagens of the predicted size were recovered from the medi
um. All were triple-helical as assayed by circular dichroism. Therefor
e, deletion of a complete D-period containing 234 amino acids does not
destabilize the triple helix of homotrimeric collagen II as much as s
ome naturally occurring mutations in the heterotrimeric monomer of col
lagen 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 e
xtracellular matrix and for the binding, spreading and signaling of ce
lls.