Proteoglycans of bovine articular cartilage. The effects of divalent cations on the biochemical properties of link protein

In cartilage proteoglycan aggregates, link protein stabilizes the binding of proteoglycan monomers to hyaluronate by binding simultaneously to hyaluronate and to the G1 globular domain of proteoglycan monomer core protein. Studies reported here involving metal chelate affinity chromatography demonstrate that link protein is a metalloprotein that binds Zn2+, Ni2+, and Co2+. Zn2+ and Ni2+ decrease the solubility of link protein and result in its precipitation. However, link protein is readily soluble and functional in low ionic strength solvents from which divalent cations have been removed with Chelex 100. These observations make it possible to study the biochemical properties of link protein in low ionic strength, physiologic solvents. Studies were carried out to define the oligomeric state of link protein alone in physiologic solvents, and the transformation in oligomeric state that occurs when link protein binds hyaluronate. Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7, link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 x 10(27) M-5, yielding a delta G' of -36 kcal/mol for the formation of the hexamer from six monomers. On binding hyaluronate oligosaccharides (HA10 or HA12), link protein dissociates to dimer. Link protein hexamer is rendered insoluble by Zn2+. Greater than 90% of the protein is precipitated by 2 mol of Zn2+/mol of link protein monomer. The binding of hyaluronate oligosaccharide by link protein strongly inhibits the precipitation of link protein by Zn2+. The link protein/hyaluronate oligosaccharide complex is completely soluble in the presence of 2 mol of Zn2+/mol of link protein. At higher molar ratios of Zn2+/link protein, the inhibitory effect of hyaluronate oligosaccharide on the precipitation of link protein is gradually overcome. Hyaluronate oligosaccharide is not dissociated from link protein by Zn2+. Hyaluronate remains bound to the link protein which is precipitated by Zn2+, or to the link protein which binds to Zn2(+)-charged iminodiacetate-Sepharose columns. Hyaluronate oligosaccharides and Zn2+ bind to different sites on link protein.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage.

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.     

Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering

The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an immunoglobulin (Ig) fold domain and two proteoglycan tandem repeat (PTR) domains. Both proteins were investigated by neutron and synchrotron X-ray solution scattering, in which nonspecific aggregate formation was reduced by the use of citraconylation to modify surface lysine residues. The neutron and X-ray radius of gyration RG of native and citraconylated binding region is 5.1 nm, and the cross-sectional RG (RXS) is 1.9-2.0 nm. No neutron contrast dependence of the RG values was observed; however, a large contrast dependence was seen for the RXS values which is attributed to the high carbohydrate content of the binding region. The neutron RG for citraconylated link protein is 2.9 nm, its RXS is 0.8 nm, and these data are also independent of the neutron contrast. The scattering curves of binding region and link protein were modeled using small spheres. Both protein structures were defined initially by the representation of one domain by a crystal structure for a variable Ig fold and a fixed volume for the two PTR domains calculated from sequence data. The final models showed that the different dimensions and neutron contrast properties of binding region compared to link protein could be attributed to an extended glycosylated C-terminal peptide with extended carbohydrate structures in the binding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin

Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.     

Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages

We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7-7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.     

Effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in cartilage explants.

The effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in calf cartilage explants were examined. Pulse-chase experiments showed that conversion of low-affinity monomers to the high-affinity form (that is, to a form capable of forming aggregates with 1.6% hyaluronate on Sephacryl S-1000) occurred with a t1/2 of about 5.7 h in free-swelling discs at pH 7.45. Static compression during chase (in pH 7.45 medium) slowed the conversion, as did incubation in acidic medium (without compression). Both effects were dose-dependent. For example, the t1/2 for conversion was increased to about 11 h by either (1) compression from a thickness of 1.25 mm to 0.5 mm or (2) medium acidification from pH 7.45 to 6.99. Oscillatory compression of 2% amplitude at 0.001, 0.01, or 0.1 cycles/s during chase did not, however, affect the conversion. Changes in the hyaluronate-binding affinity of [35S]proteoglycans in these experiments were accompanied by no marked change in the high percentage (approximately 80%) of monomers which could form aggregates with excess hyaluronate and link protein. Since static tissue compression would result in an increased matrix proteoglycan concentration and thereby a lower intra-tissue pH [Gray, Pizzanelli, Grodzinsky & Lee (1988) J. Orthop. Res. 6, 777-792], it seems likely that matrix pH may influence proteoglycan aggregate assembly by an effect on the hyaluronate-binding affinity of proteoglycan monomer. Such a pH mechanism might have a physiological role, promoting proteoglycan deposition in regions of low proteoglycan concentration.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

The affinity of newly synthesized proteoglycan for hyaluronic acid can be enhanced by exposure to mild alkali.

The affinities for hyaluronic acid of newly synthesized proteoglycan from post-confluent rabbit chondrocyte cultures and purified bovine proteoglycan monomer were compared. In mixtures prepared at pH 6.8 the newly synthesized proteoglycan had the lower affinity; however, in mixtures incubated at pH 8.5 for 24 h before addition of hyaluronic acid, the newly synthesized proteoglycan exhibited a markedly higher affinity than the bovine monomer. The results suggest that proteoglycan secreted without associated link protein [Plaas, Sandy & Muir (1983) Biochem. J. 214, 855-864] has a low affinity for hyaluronate and that this may be increased during subsequent extracellular processing.     

The interaction of link proteins with proteoglycan monomers in the absence of hyaluronic acid

Cartilage proteoglycan aggregates contain three classes of interacting components: proteoglycan monomers, hyaluronic acid and link proteins. Direct evidence is presented for a link protein to proteoglycan monomer association which hitherto has only been presumed to occur. Thus, when mixtures of purified link proteins and proteoglycan monomers were subjected to ultracentrifugation or gel chromatography under ‘associative’ conditions, link proteins were found to fractionate with proteoglycan.     

Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro.

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.     

Use of immobilized light-harvesting chlorophyll a/b protein to study the stoichiometry of its self-association.

D. J. Davis & E. L. Gross (1976) Biochim. Biophys. Acta 449, 554-564 previously observed that the light-harvesting chlorophyll a/b protein or chlorophyll protein complex II self-associated as determined by ultracentrifugation. We have determined the stoichiometry of complex formation by immobilizing the monomer on ethylenediamine-Sepharose 4B and determing the ability of immobilized protein to bind the free protein. The amount of soluble protein bound to the immobilized protein increased as the concentration of soluble protein increased. The binding was maximal between pH 7 and 8. The maximum binding was three molecules bound per one molecule of protein immobilized. These results indicate that a tetramer is the intrinsic structural unit of the light-harvesting chlorophyll a/b protein in the chloroplast membrane. Upon complex formation, the chlorophyll fluorescence was decreased without any spectral change. The maximum binding was approximately doubled upon addition of 0.5 mM CaCl2 whereas 5 mM NaCl had no effect. Addition of CaCl2 had no effect on the fluorescence of the monomer. The light-harvesting chlorophyll a/b protein can be isolated from a sodium lauryl sulfate extract of chloroplasts by affinity chromatography using the immobilized light-harvesting chlorophyll a/b protein.     

Link protein interactions with hyaluronate and proteoglycans. Characterization of two distinct domains in bovine cartilage link proteins

Hyaluronic acid-binding region and trypsin-link protein were prepared from bovine nasal cartilage proteoglycan complex after trypsin digestion. Binary complexes were reformed between trypsin-link protein and hyaluronic acid-binding region or hyaluronate. Upon trypsin treatment of these complexes, two fragments deriving from trypsin-link protein were characterized. One of them, of 20 kDa, corresponds in fact to a 140-amino acid long fragment and bears the glycosylated site of trypsin-link protein; it appears to be involved in proteoglycan/link protein interaction. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link protein with hyaluronic acid. A structural model of bovine trypsin-like protein depicting two distinct domains involved in hyaluronate and proteoglycan subunit interactions is proposed.     

Specific chemical modifications of link protein and their effect on binding to hyaluronate and cartilage proteoglycan

Specific chemical modifications of amino acid residues were performed on purified, native link protein from bovine articular cartilage. The effects of these on link protein's interactions with hyaluronate and bovine articular cartilage proteoglycan were assayed by gel chromatography. Interaction with hyaluronate was significantly perturbed by modification of lysine, arginine, tyrosine and aspartic/glutamic acid residues, but not histidine and tryptophan residues. No free, accessible sulphydryl group was found on native link protein. The requirement for unmodified lysine and arginine residues resembles that of the hyaluronate-binding site of pig laryngeal cartilage proteoglycan (Hardingham, T.E., Ewins, R.J.F. and Muir, H. (1976) Biochem. J. 157, 127-143). In contrast, proteoglycan binding was only significantly perturbed by the loss of arginine residues. This resistance may reflect hydrophobicity of the binding site or masking of the site from chemical modification by link protein self-association. Amidation of carboxyl groups, which destroyed hyaluronate binding but left proteoglycan binding intact, provides a means of generating a monofunctional link protein molecule of potential use in proteoglycan aggregation studies.     

Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein.

The N-terminal fragment (G1-G2) of cartilage proteoglycan protein core contains two globular domains, binding region (G1) and a second globular domain (G2), G1-G2 was isolated after mild trypsin digestion of purified proteoglycan aggregates followed by chromatography first on Sepharose CL-2B under associative conditions and then on a TSK-4000 column in 4 M-guanidinium chloride. It migrated as a single band (apparent Mr 150,000) on SDS/polyacrylamide-gel electrophoresis. G2 was isolated by V8-proteinase digestion of G1-G2 followed by aggregation of the G1-containing fragments with hyaluronate and chromatography on TSK-4000. It migrated as a single band on SDS/polyacrylamide-gel electrophoresis of apparent Mr 66,000 after digestion with keratanase. G2 did not interact with proteoglycan monomer, hyaluronate, link protein or other extractable cartilage matrix proteins. A polyclonal antibody raised against G2 did not cross-react with G1 or link protein. These data show that, despite a high degree of sequence similarity, G1 and G2 do not share any functional properties nor have major antigenic sites in common.     

A study of equilibrium binding of link protein to hyaluronate.

Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.