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A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

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Repeat III2 contains a FN binding site. Solid phase binding assays were used to examine binding of FN (A–C) and NH2- terminal 70-kD fragment (B) to immobilized fusion proteins. (A and C) MBP and fusion proteins were coated at 2 μg/ml and incubated with 50 μg/ml rat pFN. (B) MBP-III2 coated at 4 μg/ml was incubated with rat pFN or 70-kD fragment at increasing concentrations. Background binding to MBP was subtracted from these data. Binding was detected with 5G4 anti-FN monoclonal antibody for FN (A and C) or R457 anti–70-kD polyclonal antiserum (B). Results are presented as the average of normalized values from two to three experiments.
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fig7: Repeat III2 contains a FN binding site. Solid phase binding assays were used to examine binding of FN (A–C) and NH2- terminal 70-kD fragment (B) to immobilized fusion proteins. (A and C) MBP and fusion proteins were coated at 2 μg/ml and incubated with 50 μg/ml rat pFN. (B) MBP-III2 coated at 4 μg/ml was incubated with rat pFN or 70-kD fragment at increasing concentrations. Background binding to MBP was subtracted from these data. Binding was detected with 5G4 anti-FN monoclonal antibody for FN (A and C) or R457 anti–70-kD polyclonal antiserum (B). Results are presented as the average of normalized values from two to three experiments.

Mentions: To demonstrate a requirement for III2 in FN assembly, attempts were made to generate a recFN lacking repeat III2 or with another homologous repeat in its place. Unlike the recFNs reported here, secretion of mutant recFNs lacking III2 from infected insect cells was very inefficient, suggesting that in the absence of this repeat the proteins were not properly folded. We have shown previously that III1–2 purified in soluble form from bacterial lysates is able to bind FN (Aguirre et al., 1994). To identify the repeat responsible for FN binding activity, maltose-binding protein (MBP) fusion proteins containing either III1 or III2 were generated and tested for binding in solid phase binding assays. MBP-III2, but not MBP-III1, showed significant FN binding activity (Fig. 7 A) that was reversed by treatment with buffered SDS (unpublished data). The lack of MBP-III1 binding confirms the results of Ingham et al. (1997) who showed that native III1 does not bind to FN or its fragments. FN binding was concentration dependent and a complementary binding site was localized to the 70-kD region (Fig. 7 B). Apparent dissociation constants for FN and 70 kD binding to III2 differ by only 3.5-fold, 28 nM, and 8 nM, respectively. The higher dissociation constant for FN may reflect a difference in affinity. However, it may also be due to molecular differences between the relatively small 70-kD fragment and dimeric pFN, which has a compact conformation that may reduce accessibility to III2 binding sites.


A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

Repeat III2 contains a FN binding site. Solid phase binding assays were used to examine binding of FN (A–C) and NH2- terminal 70-kD fragment (B) to immobilized fusion proteins. (A and C) MBP and fusion proteins were coated at 2 μg/ml and incubated with 50 μg/ml rat pFN. (B) MBP-III2 coated at 4 μg/ml was incubated with rat pFN or 70-kD fragment at increasing concentrations. Background binding to MBP was subtracted from these data. Binding was detected with 5G4 anti-FN monoclonal antibody for FN (A and C) or R457 anti–70-kD polyclonal antiserum (B). Results are presented as the average of normalized values from two to three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196193&req=5

fig7: Repeat III2 contains a FN binding site. Solid phase binding assays were used to examine binding of FN (A–C) and NH2- terminal 70-kD fragment (B) to immobilized fusion proteins. (A and C) MBP and fusion proteins were coated at 2 μg/ml and incubated with 50 μg/ml rat pFN. (B) MBP-III2 coated at 4 μg/ml was incubated with rat pFN or 70-kD fragment at increasing concentrations. Background binding to MBP was subtracted from these data. Binding was detected with 5G4 anti-FN monoclonal antibody for FN (A and C) or R457 anti–70-kD polyclonal antiserum (B). Results are presented as the average of normalized values from two to three experiments.
Mentions: To demonstrate a requirement for III2 in FN assembly, attempts were made to generate a recFN lacking repeat III2 or with another homologous repeat in its place. Unlike the recFNs reported here, secretion of mutant recFNs lacking III2 from infected insect cells was very inefficient, suggesting that in the absence of this repeat the proteins were not properly folded. We have shown previously that III1–2 purified in soluble form from bacterial lysates is able to bind FN (Aguirre et al., 1994). To identify the repeat responsible for FN binding activity, maltose-binding protein (MBP) fusion proteins containing either III1 or III2 were generated and tested for binding in solid phase binding assays. MBP-III2, but not MBP-III1, showed significant FN binding activity (Fig. 7 A) that was reversed by treatment with buffered SDS (unpublished data). The lack of MBP-III1 binding confirms the results of Ingham et al. (1997) who showed that native III1 does not bind to FN or its fragments. FN binding was concentration dependent and a complementary binding site was localized to the 70-kD region (Fig. 7 B). Apparent dissociation constants for FN and 70 kD binding to III2 differ by only 3.5-fold, 28 nM, and 8 nM, respectively. The higher dissociation constant for FN may reflect a difference in affinity. However, it may also be due to molecular differences between the relatively small 70-kD fragment and dimeric pFN, which has a compact conformation that may reduce accessibility to III2 binding sites.

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

Show MeSH