Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.     

In vitro collagen fibril assembly in glycerol solution: evidence for a helical cooperative mechanism involving microfibrils

Glycerol inhibits the in vitro self-association of monomeric collagen into fibrils and induces the dissociation of fibrils preassembled from NaBH4-reduced collagen. These effects were investigated in an effort to understand the mechanism of fibril assembly of the protein. In PS buffer (0.03 M NaPi and 0.1 M NaCl, pH 7.0) containing 0.1-1.0 M glycerol, the self-association of type I collagen from calf skin took place only if the protein concentration was above a critical value. This critical protein concentration increased with increasing glycerol concentration. Velocity sedimentation studies showed that below the critical protein concentration and under fibril assembly conditions, the collagen was predominantly in a monomeric state. Electron microscopic examinations revealed that the collagen aggregates formed above the critical concentration consisted mostly of microfibrils of 3-5-nm diameter along with some banded fibrils were found. Collagen treated with pepsin to remove its nonhelical telopeptides also self-associated into microfibrils and fibrils in the presence of glycerol, but the reaction did not exhibit any critical concentration. These results are consistent with a mechanism of in vitro collagen fibril assembly which involves the initial formation of microfibrils through a helical cooperative mechanism. They also suggest that contacts of the nonhelical telopeptides of each collagen with its neighboring molecules provide the necessary negative free energy change for the cooperativity and that subsequent lateral association of the microfibrils leads to banded fibrils.     

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.     

Attachment of cells to basement membrane collagen type IV

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.     

Interaction of DNA with connective tissue matrix proteins reveals preferential binding to type V collagen

The interaction of DNA with type I to VI collagens and laminin was studied in vitro in systems in which the connective tissue components were immobilized, as well as when in solution. In studies on immobilized components, significant binding of DNA was observed only for type V collagen, and the binding of radiolabeled DNA to this component could be effectively inhibited in a concentration-dependent manner by the addition of unlabeled DNA. Similar results were observed in solution assays in which it was observed that DNA binding to type V collagen was dependent on the native triple-helical conformation of the collagen. The preferential binding of DNA to native type V collagen may be due to the relative basicity of type V collagen chains, as well as the unique spatial arrangement of amino acid side chains in the native molecules. The data are of potential clinical relevance in that binding of DNA to type V collagen may represent at least one component of the mechanism whereby DNA and its immune complexes are deposited in connective tissues in certain pathologic conditions.     

Interactions between Cells and Collagen V Molecules or Single Chains Involve Distinct Mechanisms

Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the α chains by heparin affinity chromatography. Isolated α2(V) chains, rich in RGD sequences, were more efficient than isolated α1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for α1α1 and α2β1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Single strand DNA binding of simian virus 40 tumor antigen.

Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.     

Equilibrium thermal transitions of collagen model peptides

The folding of collagen in vitro is very slow and presents difficulties in reaching equilibrium, a feature that may have implications for in vivo collagen function. Peptides serve as good model systems for examining equilibrium thermal transitions in the collagen triple helix. Investigations were carried out to ascertain whether a range of synthetic triple-helical peptides of varying sequences can reach equilibrium, and whether the triple helix to unfolded monomer transition approximates a two-state model. The thermal transitions for all peptides studied are fully reversible given sufficient time. Isothermal experiments were carried out to obtain relaxation times at different temperatures. The slowest relaxation times, on the order of 10-15 h, were observed at the beginning of transitions, and were shown to result from self-association limited by the low concentration of free monomers, rather than cis-trans isomerization. Although the fit of the CD equilibrium transition curves and the concentration dependence of T(m) values support a two-state model, the more rigorous comparison of the calorimetric enthalpy to the van't Hoff enthalpy indicates the two-state approximation is not ideal. Previous reports of melting curves of triple-helical host-guest peptides are shown to be a two-state kinetic transition, rather than an equilibrium transition.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.     

Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction.

The denatured alpha1(I) chain and the cyanogen bromide peptide, alpha1(I)-CB5, of chick skin collagen cause the relaese of serotonin and leakage of lactic dehydrogenase from human platelets in a manner similar to the release reaction mediated by adenosine diphosphate and native collagen. These peptides also cause a decrease in the level of adenosine 3':5'-monophosphate (cAMP) in platelets. Adenylate cyclase activity of platelets is partially inhibited by these peptides as well as by native collagen, ADP, and epinephrine, but cAMP phosphodiesterase activity is unaltered by these substances. In contrast, the level of platelet guanosine 3':5'-monophosphate (cGMP) is increased by the collagen peptides as well as the other aggregating agents. The increase is associated with increased guanylate cyclase, but normal cGMP phosphodiesterase activities of platelets. Optical rotatory and viscometric measurements of the alpha1 chains and alpha1-CB5 of chick skin in 0.01 M phosphate/0.15 M sodium chloride, pH 7.4, at various temperatures as a function of time indicate that no detectable renaturation occurs at 37 degrees for at least 30 min of observation. Molecular sieve chromatography of alpha1-CB5 in the phosphate buffer at 37 degrees shows that its elution position is identical to that performed under denaturing conditions (at 45 degrees) with no evidence of higher molecular weight aggregates, and the alpha1-CB5 glycopeptide fraction eluting from the column at the position of its monomer retains the platelet aggregating activity. Additionally, electron microscopic examination of the platelet-rich plasma that had been reacted with these peptides fail to show any ordered collagen structures. These data indicate that the denatured alpha1 chain and alpha1-CB5 glycopeptide of chick skin collagen mediate platelet aggregation through the "physiologic" release reaction in a manner similar to that induced by other aggregating agents such as ADP, epinephrine, or native collagen, and support the conclusion that the aggregating activity of the alpha1 chain and alpha1-CB5 is not likely to be due to the formation of polymerized products.     

Template-assembled triple-helical peptide molecules: mimicry of collagen by molecular architecture and integrin-specific cell adhesion

Most proteins fold into specific structures to exert their biological functions, and therefore the creation of protein-like molecular architecture is a fundamental prerequisite toward realizing a novel biologically active protein-like biomaterial. To do this with an artificial collagen, we have engineered a peptide template characterized by its collagen-like primary structure composed of Gly-Phe-Gly-Glu-Glu-Gly sequence to assemble (Pro-Hyp-Gly)n (n = 3 and 5) into triple-helical conformations that resemble the native structure of collagen. The peptide template has three carboxyl groups connected to the N-termini of three collagen peptides. The coupling was accomplished by a simple and direct branching protocol without complex strategies. A series of biophysical studies, including melting curve analyses and CD and NMR spectroscopy, demonstrated the presence of stable triple-helical conformation in the template-assembled (Pro-Hyp-Gly)3 and (Pro-Hyp-Gly)5 solution. Conversely, nontemplated peptides showed no evidence of assembly of triple-helical structure. A cell binding sequence (Gly-Phe-Hyp-Gly-Glu-Arg) derived from the collagen alpha1(I) chain was incorporated to mimic the integrin-specific cell adhesion of collagen. Cell adhesion and inhibition assays and immunofluorescence staining revealed a correlation of triple-helical conformation with cellular recognition of collagen mimetics in an integrin-specific way. This study offers a robust strategy for engineering native-like peptide-based biomaterials, fully composed of only amino acids, by maintaining protein conformation integrity and biological activity.     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.     

Bindings of RNAse to denatured and native deoxyribonucleic acids

The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 10⁴ l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA.