Gelatin binding to the 6F1(1)F2(2)F2 fragment of fibronectin is independent of module-module interactions

Fibronectin, a large modular protein, interacts with many other proteins in the extracellular matrix and on the cell surface. It has previously been shown that interactions between noncontiguous modules exist in the collagen binding region. It is shown here that the interaction between the sixth type I module ((6)F1) and the second type II module ((2)F2) can be disrupted by mutation of a residue in the intermodule interface of the (6)F1(1)F2(2)F2 fragment. The perturbation of the interface and the binding of collagen-derived peptides to individual modules were assessed by high-resolution nuclear magnetic resonance (NMR) spectroscopy. Cooperativity between the modules in binding ligand was assessed by analytical gelatin affinity chromatography of the mutant and wild-type proteins. Differential scanning calorimetry (DSC) was used to probe the influence of the interface on module stability. It is shown that while the (6)F1-(2)F2 interface confers significant thermal stability to the (2)F2 module, it has little effect on gelatin binding activity of the (6)F1(1)F2(2)F2 fragment.     

Solution structure of the N-terminal F1 module pair from human fibronectin.

Multiple sites within the N-terminal domain (1-5F1) of fibronectin have been implicated previously in fibronectin matrix assembly, heparin binding, and binding to cell surface proteins of pathogenic bacteria. The solution structure of 1F1(2)F1, the N-terminal F1 module pair from human fibronectin, has been determined using NMR spectroscopy. Both modules in the pair conform to the F1 consensus fold. In 4F1(5)F1, the only other F1 module pair structure available, there is a well-defined intermodule interface; in 1F1(2)F1, however, there is no detectable interface between the modules. Comparison of the backbone 15N-{1H} NOE values for both module pairs confirms that the longer intermodule sequence in 1F1(2)F1 is flexible and that the stabilization of the 4F1 C-D loop observed in 4F1(5)F1, as a result of the intermodule interface, is not observed in 1F1(2)F1.     

Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA

Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules ((5)F1 and (6)F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2- or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes (1-5)F1 and lacks modules from the adjacent gelatin-binding domain. However, when only (1-3)F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of (1-5)F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment.     

Peptide ligands for the fibronectin type II modules of matrix metalloproteinase 2 (MMP-2)

The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQFPAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mm(-1) range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction.     

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.     

Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

The relative orientation of the fibronectin 6F11F2 module pair: A 15N NMR relaxation study

The structure of a pair of modules (⁶F1¹F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from ¹H-¹H NOE and ³ J _HHN data [Bocquier et al. (1999) Structure, 7, 1451–1460] and a weak module–module interface, comprising Leu19 and Leu28, in ⁶F1, and Tyr68 in ²F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of ¹⁵N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and ³ J _HHN data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where ¹⁵N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443–449].     

Solution structure of a pair of modules from the gelatin-binding domain of fibronectin

BACKGROUND: Fibronectin has a role in vital physiological processes such as cell migration during embryogenesis and wound healing. It mediates the attachment of cells to extracellular matrices that contain fibrous collagens. The affinity of fibronectin for native collagen and denatured collagen (gelatin) is located within a 42 kDa domain that contains four type 1 (F1) and two type 2 (F2) modules. A putative ligand-binding site has been located on an isolated F2 module, but the accessibility of this site in the intact domain is unknown. Thus, structural studies of module pairs and larger fragments are required for a better understanding of the interaction between fibronectin and collagen. RESULTS: The solution structure of the 101-residue 6F1 1F2 module pair, which has a weak affinity for gelatin, has been determined by multidimensional NMR spectroscopy. The tertiary structures determined for each module conform to the F1 and F2 consensus folds established previously. The experimental data suggest that the two modules interact via a small hydrophobic interface but may not be tightly associated. Near-random-coil 1H NMR chemical shifts and fast dynamics for backbone atoms in the linker indicate that this region is unlikely to be involved in the overall stabilisation of the module pair. CONCLUSIONS: The modules in the 6F1 1F2 module pair interact with each other via a flexible linker and a hydrophobic patch, which lies on the opposite side of the 1F2 module to the putative collagen-binding site. The intermodule interaction is relatively weak and transient.     

Identification of residues involved in the interaction of Staphylococcus aureus fibronectin-binding protein with the (4)F1(5)F1 module pair of human fibronectin using heteronuclear NMR spectroscopy

Many pathogenic Gram-positive bacteria express cell surface proteins that bind to components of the extracellular matrix. This paper describes studies of the interaction between ligand binding repeats (D3 and D1-D4) of a fibronectin-binding protein from Staphylococcus aureus with a module pair ((4)F1(5)F1) from the N-terminal region of fibronectin. When D3 was added to isotope-labeled (4)F1(5)F1, (1)H, (15)N, and (13)C NMR chemical shift changes indicate that binding is primarily via residues in (4)F1, although a few residues in (5)F1 are also affected. Both hydrophobic and electrostatic interactions appear to be involved. The NMR data indicate that part of the D3 repeat converts from a disordered to a more ordered, extended conformation on binding to (4)F1(5)F1. In further NMR experiments, selective reduction of the intensity of D1-D4 resonances was observed on binding to (4)F1(5)F1, consistent with previous suggestions that in each of D1, D2, and D3 repeats, the main fibronectin binding site is in the C-terminal region of the repeat. In D1-D4, these regions also appear to go from a disordered to a more ordered conformation of fibronectin binding. Although the regions of the two proteins which interact had been previously identified, the findings presented here identify, for the first time, the specific residues in both proteins that are likely to be involved in the interaction.     

Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin

Background

Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules ⁷F3-¹⁰F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules ⁷F3-¹⁰F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly.

Methodology/Principal Findings

To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to ⁷F3-¹⁰F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules ²F3-¹⁴F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module ¹F3 or the C-terminal modules to modules ²F3-¹⁴F3 resulted in some activity, and addition of both ¹F3 and the C-terminal modules resulted in a construct, ¹F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs ¹F3-C V0, ¹F3-C V64, and ¹F3-C Δ(V¹⁵F3¹⁰F1) were all able to support fibronectin assembly, suggesting that ¹F3 through ¹¹F1 and/or ¹²F1 were important for activity. Coatings in which the active parts of ¹F3-C were present in different proteins were much less active than intact ¹F3-C.

Conclusions

These results suggest that ¹F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.     

Integrity of refolded and reoxidized gelatin-binding fragments of fibronectin.

The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.     

Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.     

Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules

Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.     

The solution and crystal structures of a module pair from the Staphylococcus aureus-binding site of human fibronectin--a tale with a twist

An important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type 1 modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV radiation damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins.     

Mutations in the beta-subunit Thr(159) and Glu(184) of the Rhodospirillum rubrum F(0)F(1) ATP synthase reveal differences in ligands for the coupled Mg(2+)- and decoupled Ca(2+)-dependent F(0)F(1) activities

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.     

Folding and stability of the b subunit of the F(1)F(0) ATP synthase

The F(1)F(0) ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F(0)) and soluble (F(1)) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F(1)F(0) complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T(m) values between 30 and 40 degrees C. CD spectra for the region comprising residues 53-122 (b(53-122)) showed theta;(222)/theta;(208) = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b(53-122) (DeltaG, DeltaH and DeltaC(p)) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.     

Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin

Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.     

All six modules of the gelatin-binding domain of fibronectin are required for full affinity

The gelatin-binding sites of fibronectin are confined to a 42-kDa region having four type I and two type II modules in the following order: I(6)-II(1)-II(2)-I(7)-I(8)-I(9). To determine the relative importance of each module for recognition of gelatin, recombinant green fluorescent fusion proteins were prepared in which individual modules or groups of modules were deleted, and the resulting proteins were tested for binding to gelatin by analytical affinity chromatography. Deletion of both type II modules did not eliminate binding, confirming that at least some of the type I modules in this region are able to bind gelatin. It was found that deletion of type I module 6 tends to increase the affinity, whereas deletion of any other module decreases it. Deletion of module I(9) had a large effect but only if module II(2) was also present, suggesting an interaction between these two noncontiguous modules. Analysis of more than 20 recombinant fusion products led to the conclusion that all modules contribute to the interaction either directly by contacting the ligand or indirectly through module-module interactions.     

F2breed,a New Program for Construction of Genetic Linkage Maps

This paper presents a new program F2breed for Windows and Linux operating systems, which was designed for estimation of recombination frequency between the genetic loci and construction of genetic maps based on the analysis of inheritance in a F2 population. The program implements the approach of the orientation of n points corresponding to n analyzed loci in the (n–1)-dimensional space for relative arrangement of the loci on the genetic map by consideration of various projections of this structure. The program is characterized by a convenient, intuitive interface, is easy to operate, and is suitable for working with small groups of loci (up to 100).