The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Copper staining method for extracting biologically active proteins from native gels.

An attempt was made to make protein bands visible on native gel using copper staining, since such a mild staining procedure would make the entire native gel electrophoresis process non-denaturing. Copper staining not only was able to detect various proteins on native gel with reasonable sensitivity, but also made extraction and recovery of active proteins possible from the gel using a gentle procedure.

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

Fractionation and characterization of polyclonal antibodies using three progressively more chaotropic solvents.

In the previous paper we described the effect of several different solvents on the structure of antibodies and demonstrated that 0.1 M glycine, pH 2.9, 7 M urea, pH 4.0, and 6 M guanidine-HCl, pH 4.0, unfold the antibodies to different degrees. Antibodies can be refolded from all of these solvents by dialysis. Polyclonal antibodies (pAbs) are a mixture of antibodies which recognize and bind different epitopes on the same antigen, with the strength of the antigen-antibody binding varying with each subpopulation. When rabbit antisera to the extracellular domain of Her2 receptor (sHer2), derived from Chinese hamster ovary cells, was applied to an antigen column, bound pAbs were recovered with a step-wise elution of 0.1 M glycine, pH 2.9 (44% of the total recovered pAb), 7 M urea, pH 4.0 (29%), and 6 M guanidine-HCl, pH 4.0 (27%), with baseline resolution between them. Fluorescence spectra of the pAbs confirmed that the 0. 1 M glycine pH 2.9 sample had near-native structure, the pAbs in 7 M urea, pH 4.0, were partially unfolded, and the pAbs in the 6 M guanidine-HCl, pH 4.0, were totally unfolded. The glycine- or urea-eluted sample was refolded by dialysis into PBS, while the guanidine-HCl-eluted sample was first dialyzed into the 7 M urea pH 4.0 buffer and then into PBS. The refolded material from glycine or urea had native-like spectra, while the spectrum of the protein refolded from 6 M guanidine-HCl was slightly perturbed. All three of these subpopulations of pAbs formed antigen-antibody complexes which could be isolated by gel-filtration chromatography, precipitated sHer2 during immunoprecipitation, and recognized sHer2 in Western blots. The guanidine-HCl-eluted material was most sensitive for Western blotting. Identical results were obtained with pAbs applied either in the batch mode or to the top of the column, indicating that antibody aggregation which may occur when applied from the top of the column is not responsible for the distribution of pAbs into different subpopulations. These results indicate that the sequential use of these three increasingly chaotropic solvents to elute antibodies results in both increased recovery of antibodies and fractionation of pAbs into subpopulations with potentially different antigen binding characteristics.

Dimerization of granulocyte-colony stimulating factor receptor: the Ig plus CRH construct of granulocyte-colony stimulating factor receptor forms a 2:2 complex with a ligand.

We have previously shown that the extracellular domain of granulocyte-colony stimulating factor receptor (soluble G-CSFR), prepared from CHO cell conditioned media, dimerizes upon binding its ligand, G-CSF. The most stable ligand-receptor complex occurs at a 2:2 stoichiometry, unlike the growth hormone and erythropoietin systems. In the latter cases, each ligand uses two sites to bring two receptors together. In this study, we have generated a truncated G-CSF receptor, known to be sufficient for high affinity ligand binding, which consists of an Ig-like domain and a cytokine receptor homology module. With an affinity purified receptor, sedimentation equilibrium experiments clearly demonstrated that this truncated form of the receptor behaves very similarly to the entire extracellular domain. The sedimentation equilibrium data are consistent with the model that the truncated receptor has a weak tendency to self-associate into a dimer in the absence of a ligand, this receptor-receptor interaction is enhanced by ligand binding, and the most stable complex occurs at a 2:2 stoichiometry. These results are very different from those described by others for various murine G-CSF receptor constructs from either Escherichia coli or insect expression systems.

Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences.

Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation.

Production and characterization of an analog of acidic fibroblast growth factor with enhanced stability and biological activity.

We have used recombinant DNA methods to produce two forms of bovine acidic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at position 93. Both forms were expressed at high levels in Escherichia coli and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dichroic and infrared spectra suggested that the proteins are similar in secondary and tertiary structures and contain little or no alpha-helical conformations. Hydrophobic interaction chromatography showed that aFGF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, suggesting that residue 93 is exposed to the solvent. Half-maximal activity in an in vitro bioassay system was reached at a 10- to 20-fold lower dose for the aFGF C47A/H93G form than for the aFGF C47A form, suggesting that alteration of this residue has an effect on the region responsible for receptor binding. Addition of 50 micrograms/ml heparin enhanced the in vitro activity of the aFGFs, reducing the half-maximal dose to approximately 100 pg/ml for both forms, comparable to that observed previously for basic FGF with or without heparin in this assay system.

Analysis of the heat-induced denaturation of proteins using temperature gradient gel electrophoresis.

A new technique, temperature gradient gel electrophoresis (TGGE), was applied to the study of heat-induced protein denaturation. The gels used contained 30 mM Borax + 75 mM boric acid, pH 8.4, and various concentrations of urea. When this technique was applied to bovine serum albumin, the protein showed discontinuous bands upon melting, indicating that the thermal transition is irreversible. The apparent melting temperature, calculated based on the relative intensity of two bands in the transition region, was 58 degrees C in 2 M urea. When the thermal denaturation of bovine serum albumin was analyzed spectroscopically, the transition was again irreversible, with an apparent transition temperature of 57 degrees C, consistent with the TGGE results. Recombinant stem cell factor, recombinant granulocyte macrophage-colony stimulating factor, and catalase were also analyzed by TGGE, indicating that the technique can be used to analyze denaturation of monomeric and multimeric proteins.

Effect of amino-terminal processing by Staphylococcus aureus V-8 protease on activity and structure of recombinant human interferon-gamma.

Treatment of recombinant human interferon-gamma (rHuIFN-gamma) with Staphylococcus aureus V-8 protease generated a transiently stable species that lacks 10 amino-terminal residues. This protein showed distinct secondary and higher-order structures with an alpha-helical content of 31%, suggesting that the secondary and tertiary structure largely remain upon removal of 10 amino-terminal residues. The antiviral activity was abolished, or greatly reduced, for this species relative to the intact protein. These results suggest an important role for the amino-terminal portion in the activity of human interferon-gamma. Since the digested protein is difficult to refold from the acid-denatured state, it was concluded that, although not essential for maintaining the core structure of the protein, the amino-terminal portion is critical for refolding the protein from acid.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.