Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins.

Aukhil, Ikramuddin; Joshi, Pallavi; Yan, Y. Z.; Erickson, Harold P.

doi:10.1016/s0021-9258(18)53809-6

Cited by 205 publications

(56 citation statements)

References 60 publications

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“…The cloning of fragments based on amplification of cDNA to begin and terminate each protein at the boundaries of the independently folding domains successfully yields stable fragments of the FN-III domains. We had successfully used a similar approach to map the cell-and heparin-binding domains of human tenascin (Aukhil et al, 1993). In the present study, by using different combinations of type-III fragments 7-12 of human FN that include or exclude the spliced repeats EIIIB and EIIIA, we show that including the EIIIB repeat enhances the adhesion and spreading of cells.…”

Section: Discussionmentioning

confidence: 59%

“…The methods used to produce and purify bacterial expression proteins were essentially the same as described previously with some modifications (Aukhil et al, 1993). The prokaryotic expression vector pET 11b (Studier et al, 1990) and its modified version pET 15b with a histidine tag to facilitate affinity chromatography were used.…”

Section: Cloning Specific Domains Of Human Fn Into Pet Expression Vectorsmentioning

confidence: 99%

“…After a 3 hour period of growth in 1 liter of LB with ampicillin, IPTG was added at a final concentration of 2 mM and the cultures were shaken at 37°C for another 3 hours. The protocol for induction and purification of the expressed proteins has been described previously (Aukhil et al, 1993). All the expressed proteins were soluble (seen in the supernatant after cell lysis and centrifugation).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Protein concentrations were determined by measuring the absorbance at 278 nm. The extinction coefficient for each protein was calculated and estimated as described previously (Aukhil et al, 1993). SDS-PAGE was carried out using 5-20% acrylamide gradient gels under reducing conditions and stained with Coomassie Blue.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…The assay was carried out as described by Aukhil et al (1993) and Joshi et al (1993). Wells of 96-well flat bottom polyvinylchloride plastic microtiter plates (Falcon 3912) were coated with 100 µl of various concentrations of the recombinant proteins in PBS overnight at 4°C.…”

Section: Gravity Cell Attachment Assaymentioning

confidence: 99%

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The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading

Hashimoto-Uoshima

Yan

Schneider

et al. 1997

Journal of Cell Science

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Fibronectin has a complex pattern of alternative splicing at the pre-mRNA level leading to the expression of different isoforms. The alternatively spliced domains EIIIB and EIIIA are known to be prominently expressed during development and wound healing. While the other spliced domain (CS-segment) is known to promote cell adhesion in a cell type specific manner, the biological functions of the spliced domains EIIIB and EIIIA are not well understood. In the present study, we have prepared expression proteins of specific domains of human fibronectin using a prokaryotic expression system and used the purified fragments to test their ability to support adhesion and spreading of cultured cells. Fragments from type-III domains #7 to #12 were prepared in various combinations to include or exclude the spliced domains EIIIB and EIIIA. The results indicate that cultured NIL fibroblasts adhere to many of the fragments tested. However, the cell adhesion and spreading are enhanced, especially at lower concentrations, to fragments including the domain EIIIB. The inclusion of domain EIIIA led to a decrease in the adhesion of cells and those that adhered did not spread well. When tested in a centrifugal cell adhesion assay, fragments including domain EIIIB resisted the detaching forces and stayed adhered. Fragments that included domain EIIIA were unable to resist the detaching centrifugal forces to the same extent as the fragments that included domain EIIIB alone. These results suggest that the spliced domain EIIIB may be serving important biological functions in enhancing cell adhesion and spreading. This is likely to be mediated by conformational effects because domain EIIIB alone neither exhibited any adhesive activity nor competed in inhibiting adhesion to fragments #7-10.

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Section: Discussionmentioning

confidence: 59%

Section: Cloning Specific Domains Of Human Fn Into Pet Expression Vectorsmentioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Gravity Cell Attachment Assaymentioning

confidence: 99%

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