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BMC Biotechnol
2016 Feb 16;16:16. doi: 10.1186/s12896-016-0247-z.
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Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation.
Yoo HY
,
Song YH
,
Foo M
,
Seo E
,
Hwang DS
,
Seo JH
.
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BACKGROUND: von Willebrand factor (VWF) is a key load bearing domain for mamalian cell adhesion by binding various macromolecular ligands in extracellular matrix such as, collagens, elastin, and glycosaminoglycans. Interestingly, vWF like domains are also commonly found in load bearing systems of marine organisms such as in underwater adhesive of mussel and sea star, and nacre of marine abalone, and play a critical load bearing function. Recently, Proximal Thread Matrix Protein1 (PTMP1) in mussel composed of two vWF type A like domains has characterized and it is known to bind both mussel collagens and mammalian collagens.
RESULTS: Here, we cloned and mass produced a recombinant PTMP1 from E. coli system after switching all the minor codons to the major codons of E. coli. Recombinant PTMP1 has an ability to enhance mouse osteoblast cell adhesion, spreading, and cell proliferation. In addition, PTMP1 showed vWF-like properties as promoting collagen expression as well as binding to collagen type I, subsequently enhanced cell viability. Consequently, we found that recombinant PTMP1 acts as a vWF domain by mediating cell adhesion, spreading, proliferation, and formation of actin cytoskeleton.
CONCLUSIONS: This study suggests that both mammalian cell adhesion and marine underwater adhesion exploits a strong vWF-collagen interaction for successful wet adhesion. In addition, vWF like domains containing proteins including PTMP1 have a great potential for tissue engineering and the development of biomedical adhesives as a component for extra-cellular matrix.
Fig. 1.
a PTMP1 structure from PDB (http://www.rcsb.org) b SDS-PAGE analysis of recombinant PTMP1. Lanes: M, protein maker; WC, whole cell sample; S, soluble fraction; IS, insoluble fraction; A, purified PTMP1 protein. c HPLC purification of recombinant PTMP1 with a reverse phase column chromatography and d MALDI-TOF analysis of a purified recombinant PTMP1
Fig. 2.
a Cell spreading and adhesion and b Cell proliferation of MC3T3-E1 cells on PTMP1-coated surface. Each value and error bar is the mean of triplicated sample and its standard deviation. (+) PTMP1 coated tissue culture polystyrene; (−) bare tissue culture polystyrene
Fig. 3.
a Real Time PCR expression analysis of Col type I of MC3T3-E1 after 3 days on both PTMP1 coated surface (PTMP1) and uncoated surface (NC) b Quantification of the band intensity of Col I normalized to GAPDH. Each value and error bar is the mean of triplicated sample and its standard deviation (statistical significance is designated by *p < 0.05)
Fig. 4.
a SEM image of type I collagen films in the presence (+) or absence (−) of PTMP1, b Senescence of MC3T3-E1 cells on Collagen film and denatured collagen film in the presence (+) or absence (−) of PTMP1. c Intensity plot of the senescence in (b). The ordinate is the intensity plot in percentage indicating the amount of senescence detected in (b). The abscissa is the four conditions shown in (b)
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