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bioRxiv · 10.1101/2022.11.10.516045

Production of recombinant heterotrimeric mini-procollagen I and homotrimeric II mini-procollagen II reveals new cleavage sites for BMP-1

Mariano, N.·Dieryckx, C.·Tessier, A.·Vincourt, J.-B.·Vadon-Le Goff, S.·Moali, C.
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Abstract

The proteolytic conversion of soluble procollagens into mature collagen monomers is a critical step to decrease their solubility and trigger collagen fibril formation. In the case of collagens I, II and III, this maturation process is driven by several extracellular metalloproteinases such as BMP-1, tolloid-like proteinases, meprin , meprin {beta}, ADAMTS-2 and ADAMTS-14 but the extensive characterization of these proteolytic events has been hampered by the lack of recombinant procollagens. We previously reported the production and partial characterization of recombinant homotrimeric proteins derived from procollagen III (mini-procollagens III) and, in this study, we describe how we have extended this previous work to the production of heterotrimeric mini-procollagen I and homotrimeric mini-procollagen II. These mini-procollagens include truncated triple helices and intact C-telopeptide and C-propeptide domains and were produced in suspension in HEK293-F cells with yields ranging from 2.5 mg/L to 10 mg/L after purification. They proved very useful tools to analyze the effect of calcium on the stability of the procollagen C-terminal region and to compare the procollagen C-proteinase activity of BMP-1 on the three major fibrillar procollagens or their ability to interact with various partners such as PCPE-1. Using mass spectrometry to map BMP-1 cleavage sites on the mini-procollagens, we confirmed all previously described sites but also revealed two additional cleavage sites in the 1 chain of procollagens I and II. This result shows that the mini-procollagen toolkit offers a broad range of perspectives to make functional studies but also possibly structural analyses or to develop drug screening assays.

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Mariano, N., Dieryckx, C., Tessier, A., Vincourt, J.-B., Vadon-Le Goff, S., Moali, C.. 2022-11-11. Production of recombinant heterotrimeric mini-procollagen I and homotrimeric II mini-procollagen II reveals new cleavage sites for BMP-1. https://doi.org/10.1101/2022.11.10.516045

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