Kenneth C. Ingham

Rapid methods for isolation of human plasma fibronectin

Simplified procedures have been developed for isolation of human plasma fibronectin by affinity chromatography on gelatin-agarose. In one method, fibronectin is eluted with 3 M urea, and this reagent is quickly removed by adsorbing the protein onto heparin-agarose, followed by 0.4 M NaCl elution. In a shorter process, fibronectin is eluted from gelatin-agarose simply by decreasing the buffer pH below 6. After lyophilization the purified protein can be readily dissolved in water. The fraction not adsorbed to gelatin can be used to purify other proteins, including factor VIII whose procoagulant activity is quantitatively recovered.

Cryptic Self-association Sites in Type III Modules of Fibronectin

The first type III module of fibronectin (Fn) contains a cryptic site that binds Fn and its N-terminal 29 kDa fragment and is thought to be important for fibril formation (Morla, A., Zhang, Z., and Ruoslahti, E. (1994) Nature 367, 193-196; Hocking, D. C., Sottile, J., and McKeown-Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191). A synthetic 31-mer peptide (NAPQ … TIPG) derived from the middle of domain III1 was also shown to bind Fn, but the site of its interaction was not determined (Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429). By affinity chromatography on peptide-agarose, we tested a set of fragments representing the entire light chain of plasma Fn. Only 40-kDa Hep-2 (III12-15) failed to bind. The concentration of urea required for peak elution of Fn and the other fragments decreased in the order Fn > 42-kDa GBF (I6II1-2I7-9) > 19-kDa Fib-2 (I10-12) > 110-kDa CBF(III2-10) > 29-kDa Fib-1 (I1-I5). Neither Fn nor any of the fragments bound immobilized intact III1, confirming the cryptic nature of this activity. In an effort to detect interactions between other Fn domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interaction detected was that of fluid phase III1 with immobilized denatured 110-kDa CBF and 40-kDa Hep-2, both of which contain type III domains. Analysis of subfragments revealed this activity to be dominated by domains III7 and III15. Fn itself did not bind to the denatured fragments. Thus, domain III1 contains two cryptic “self-association sites,” one that is buried in the core of the fold but recognizes many Fn fragments when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domains could play an important role in assembly of Fn multimers in the extracellular matrix.

Separation of Macromolecules by Ultrafiltration: Influence of Protein Adsorption, Protein-Protein Interactions, and Concentration Polarization

Ultrafiltration through microporous membranes is a well-established technique for concentrating dilute protein solutions and separating proteins from low molecular weight solutes such as salts or ethanol (Friedli et al., 1977; Guthorlein, 1977; Mercer, 1977), or from much larger particles such as cells (Colton et al., 1975). However, the use of this tool for fractionating proteins according to size has progressed less rapidly. Several difficulties can be identified: non-uniform pore size protein adsorption concentration polarization protein-protein interactions

Polyethylene glycol in aqueous solution: solvent perturbation and gel filtration studies.

Abstract As part of an investigation of the mechanism of precipitation of proteins by synthetic polymers, we measured the ability of oligomers and polymers of ethylene glycol to (a) alter the solubility of amino acids, (b) perturb the absorption spectra of aromatic amino acids, and (c) enhance the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS). All of these effects increased with increasing degree of polymerization, up to a molecular weight of about 400. This trend can be partially attributed to the diminishing influence of the terminal OH groups. However, small analogs such as dioxane and dimethoxyethane (lacking OH groups) were much less effective than the polymers, suggesting a cooperative effect between neighboring ethoxide residues in the polymer. In contrast, the effectiveness of polyethylene glycol (PEG) as a protein-precipitating agent continued to increase with increasing size; polymers of molecular weight 4000–6000 were substantially more effective than their 10-fold smaller homologues. On exclusion chromatographic columns, the synthetic polymers eluted much earlier than did proteins of comparable molecular weight. This discrepancy diminished in 6 m guanidine where the proteins eluted much earlier, reflecting their conversion to random coils. In contrast, the elution of PEG was only slightly affected, suggesting a random coil configuration even in the absence of denaturant. PEG-20M, a mixture prepared by coupling 2 mol of PEG-6000 with an epoxide, was resolved into two components by exclusion chromatography on Sephadex G-100. The fast-eluting component (the coupled product) was unusually effective in enhancing ANS fluorescence and was shown to bind the dye with K a = 5.1 × 10 3 , M −1 at 25 °C.

Precipitation of proteins with polyethylene glycol: characterization of albumin.

Abstract The factors which govern the precipitation of albumin by the synthetic polymer, polyethylene glycol (PEG), have been investigated as part of a broader program to determine the molecular basis of the protein-precipitating action of synthetic polymers. The concentration of PEG-4000 required for precipitation of albumin was minimum near the isoelectric point and was only slightly dependent on temperature between 4 and 30 °C. At pH ⩾ pI (pH 4.5), increasing the ionic strength shifted the precipitation curve to a higher concentration of PEG, with different salts having quantitatively similar effects. At pH 4, the opposite effect was observed and different salts varied in their effectiveness according to their position in the lyotropic series. Dilution of albumin shifted the midpoint of the precipitation curve at pH 4.5 to a higher concentration of PEG with no significant effect on the slope. Variation of the molecular weight of PEG from 6000 to 400 shifted the precipitation to a higher PEG concentration (percentage, w v ) and diminished the slope of the curves. Precipitation of albumin dimers required less PEG than did monomers and the separation between the precipitation curves for monomer and dimer was greater with PEG-400 than with PEG-4000. Labeling of albumin with 2.3 mol of dansyl chloride had no effect on the precipitation curve at pH 4.5. The polarization of dansyl fluorescence was measured as a function of PEG concentration to determine whether albumin self-associates prior to precipitation. The results indicate that albumin remains in a monomeric state even at a PEG concentration beyond that required to initiate precipitation, reflecting the highly cooperative nature of the precipitation process.

Allosteric Modulation of Ligand Binding to Low Density Lipoprotein Receptor-related Protein by the Receptor-associated Protein Requires Critical Lysine Residues within Its Carboxyl-terminal Domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit α2M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA·PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.

Influence of self-association of proteins on their precipitation by poly(ethylene glycol)

Abstract The influence of self-association on the precipitation of proteins by poly(ethylene glycol) (PEG) was investigated using several model self-associating proteins. These include α-chymotrypsin and chymotrypsinogen, β-lactoglobulin A and glutamate dehydrogenase. With few exceptions, conditions which promote self-association also enhance precipitation by PEG, while conditions which inhibit self-association also inhibit precipitation by PEG. Chymotrypsin and chymotrypsinogen self-associate at pH 8.5 and low ionic strength and, under these conditions, they were precipitated by moderate concentrations of PEG (10–20%, w v ). Low concentrations of salts ( m ) reversed the self-association and prevented precipitation up to 25% PEG. By contrast, salt did not prevent the precipitation of covalently cross-linked oligomers of chymotrypsinogen. In acid (pH 4.5), the effects of salts on both self-association and precipitation of these proteins were opposite to the effects at pH 8.5. Different salts were generally found to have similar effects, but SO42− was particularly effective in enhancing precipitation in acid. At pH 3 and 8, β-lactoglobulin A is primarily a monomer and was not precipitated by 25% PEG at these pH values. Maximum precipitation occurred at pH 4.7, which is the reported optimum for self-association. Glutamate dehydrogenase associates to polymers at neutral pH and was precipitated by 15% PEG. The combined presence of NADH and GTP (1 m m ) prevented polymer formation and abolished precipitation. Toluene further enhanced polymerization in the absence of cofactors but did not enhance precipitation. However, PEG was found to strongly inhibit the polymerization induced by toluene. The results of these studies suggest that it may be possible to selectively enhance or inhibit the PEG precipitation of a self-associating protein through manipulation of its oligomeric state.

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: I6-II1-II2-I7-I8-I9. 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 I9 had a large effect but only if module II2 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.

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and α1-acid glycoprotein

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity.

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.