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.     

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of alpha-lactalbumin, gamma-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of alpha-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced alpha-lactalbumin. Reduction-induced changes in the conformation of alpha-lactalbumin, in the absence and presence of clusterin, were monitored by real-time (1)H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of alpha-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of alpha-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and gamma-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein.     

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.     

Lack of effect of lysozymes on cartilage proteoglycans.

Certain similarities between lysozyme and testicular hyaluronidase suggested that the putative action of the former in disaggregation of cartilage proteoglycans might be explained by a selective effect of the enzyme on the hyaluronic acid portion of the aggregate. Human lysozyme did not reduce the viscosity of either hyaluronate or of aggregated proteoglycans, it did not reduce the sedimentation velocity of hyaluronate, and it did not alter the chromatographic profile of the aggregate in a system sensitive to the difference between aggregate and its subunit. The role of human cartilage lysozyme in disaggregation of cartilage proteoglycans remains uncertain.     

Cartilage proteoglycans. Structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate.

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.     

More than 15 years of CA 125: what is known about the antigen, its structure and its function

In 1997 CA 125 celebrated its 15th anniversary. Since the discovery of OC 125, an antibody that recognizes CA 125, by Bob Bast and his colleagues, considerable progress has been made toward the development of more sensitive and more precise assay systems. However, a great deal of mystery still remains about the CA 125 molecule and further enlightenment will probably not come until the gene for CA 125 is cloned and the complete open reading frame for the peptide core identified. In the meantime, we have learned some structural features of the CA 125 molecule as well as a little about its regulation and the requirements for its secretion or release from epithelial derived cells in cultures. The CA 125 molecule is almost certainly a glycoprotein with a predominance of O-linkages. It is heterogeneous with regard to both size and charge, most likely due to continuous deglycosylation of side chains during its life-span in bodily fluids. It exists as a very large complex (perhaps as much as 4 million daltons) under natural conditions. The core CA 125 subunit is in excess of 200,000 daltons and it retains the capacity to bind both OC 125 class antibodies and M 11 class antibodies. As a denatured purified subspecies the CA 125 molecule appears to autoproteolyse presumably due to an endogenous protease activity inherent to the molecule. Release or secretion of CA 125 appears directly linked to the epithelial growth factor receptor signal transduction pathway. Prior to its release from cultured cells, CA 125 is phosphorylated (at either/both serine and threonine) and dephosphorylated when released. To stimulate discussion on the regulation of CA 125 synthesis, its secretion and its structural configuration, we have presented a model of a theoretical CA 125 molecule. Perhaps it will provide a focus of attention until the CA 125 gene is cloned and the real molecule is described.     

Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative

Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme.     

Why do worms need cholesterol?

Cholesterol is a structural component of animal membranes that influences fluidity, permeability and formation of lipid microdomains. It is also a precursor to signalling molecules, including mammalian steroid hormones and insect ecdysones. The nematode Caenorhabditis elegans requires too little cholesterol for it to have a major role in membrane structure. Instead, its most probable signalling functions are to control molting and induce a specialized non-feeding larval stage, although no cholesterol-derived signalling molecule has yet been identified for these or any other functions.     

Activity correlations in the phorbol ester series

Activity correlations in the phorbol ester series. The production of inflammation by phorbol esters on mammalian skin correlates on a structural basis with in vitro measurements of lymphocyte mitogenesis and mobilization of prostaglandins. All of the pro-inflammatory phorbol esters tested in our laboratory have been shown to activate the enzyme protein kinase C, and such an interaction could in large part explain the induction of an inflammatory response in vivo. Certain of these compounds additionally induce aggregation of human and rabbit blood platelets. This activity does not structurally correlate with the induction of inflammation but may correlate with the known tumour-promoting actions of phorbol derivatives. Compounds which induce platelet aggregation stimulate the secretion of a biologically active substance which we have termed 'Factor W'. The production of Factor W occurs into human plasma following platelet stimulation by phorbol tumour-promoting agents. It is an unstable substance, distinct in its aggregating properties from phorbol esters, ADP, 5-hydroxytryptamine, thrombin, platelet aggregating factor and the products of arachidonate oxidative metabolism.     

Identification of the core structure of lysozyme amyloid fibrils by proteolysis

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.     

Periodate oxidation and the shapes of glycosaminoglycuronans in solution.

1. It is proposed that periodate oxidation of glycol groups in the repeating units of polysaccharide molecules can be used to probe differences in polymer shapes in solution. 2. Measurement of second-order rate constants (k2) of periodate-glycol reactions may be compared between polymers and relevant monomers, to assess perturbations due to polymer configuration. 3. Factors effecting the measurement and interpretation of k2 are discussed. Over-oxidation, free-radical side reactions, end-group effects, Donnan equilibria and polymer (or molecular-weight) effects are relevant, but their importance is either small or can be minimized in practice. 4. A small group of glycosaminoglycuronans (chondroitin 4- and 6-sulphates and hyaluronate) are oxidized 50--100 times more slowly than three other glycosaminoglycuronans of similar composition, relevant monomers or three homopolyuronides. 5. A stable configuration in solution is postulated for the periodate-resistant polymers, involving carboxylate, acetamido and hydroxy groups in hydrogen-bonded sequences on alternate sides of the molecule. The more easily oxidizable polyuronides are unable to form this configuration. 6. The effect of temperature on the postulated configuration is investigated through the Arrhenius plot of k2, measured to hyaluronate, chondroitin 6-sulphate and methyl 4-O-methyl-alpha-D-glucopyranoside. Probable transitions at high (around 90 degrees C) temperatures were observed for both polymers, with an additional transition at about 37 degrees C in the case of hyaluronate. 7. L-Iduronic acid can take up different conformations depending on the polymer environment.     

Structural requirements for the interaction between peptide antigens and I-Ed molecules

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.     

A minimum-surface mechanism to account for the organization of cells into columns in the mammalian epidermis.

It has recently been discovered that the stratum corneum and superficial living epidermis of mammalian skin are organized into neat vertical columns of interdigitating cells. The mechanism for this organization has not yet been determined. This study shows that the stacked organization is not unique to the epidermis but also occurs in cork cambium and the pith of woody plant stems. The structural and spatial organization of the stacked cells in these tissues were compared and found to approximate closely the shape of Kelvin's minimum-surface polygon, the tetrakaidecahedron. Paper models of flattened tetrakaidecahedra were constructed and found to stack columns of interdigitating units which are consistent with all of the structural details seen in stacked cells observed under the light and the scanning electron microscopes. Indeed, only polygons of this type are capable of aggregating without interstices in the manner of the stacked cells. A study of stacked arrays of these models has revealed a mechanism by which the epidermis might become organized in columns of stacked and interdigitating cells. The proposed mechanism assumes that cells within an array seek the smallest possible surface-to-volume ratio and, under appropriate conditions, form stacked tetrakaidecahedra.     

Lysozyme in preosseous cartilage VI. Purification, characterization and localization of mammalian cartilage lysozyme

Lysozyme (mucopeptide-N-acetylmuramylhydrolase, EC 3.2.1.17) is present in mammalian cartilage. Lysozyme was isolated and purified from bovine and canine cartilage and from dog serum using various chromatographic steps and affinity chromatography on carboxymethylated chitin. Amino acid analysis of bovine cartilage lysozyme showed that it is similar to other mammalian lysozymes. Anti-canine lysozyme antibodies cross-react with calf lysozyme, but not with hen egg white or embryonic chick cartilage lysozyme. In the epiphyseal plate of the dog, 90-μm sections were analyzed for lysozyme and its was found that in the hypertrophic zone its concentration is approximately six times higher than it is in the resting zone. Using immunocytochemical techniques at the electromicroscopic level, lysozyme in the epiphyseal plate of the dog was localized extracellularly, mainly in the immediate vicinity of the chondrocytes, the territorial matrix.     

Effects of chemical disruption on the biological activities of sea urchin egg jelly

In an attempt to relate the biological activities of sea urchin egg jelly to the structural characteristics of the acid glycoprotein molecule, the jelly was oxidized with H₂O₂ and sodium periodate, and digested with trypsin and pronase. The non-dialyzable products of H₂O₂ and periodate oxidation, and a fucose-rich fraction isolated from enzyme-digested jelly by column chromatography, were tested for their capacity to induce sperm agglutination and acrosome reaction in Hemicentrotus pulcherrimus. It was found that a degree of enzyme digestion sufficient to remove about 80% of the amino acids reduced, but failed to eliminate, the capacity of the jelly to elicit agglutination and acrosomal reaction. Mild oxidation with H₂O₂ suppressed sperm agglutination, but more drastic treatment was required to destroy the capacity of the jelly to induce the acrosome reaction. The loss of both these biological activities after periodate oxidation was found to parallel the release of sialic acid.     

Influence of protein molecule conformation on the character of its interaction with a phospholipid bilayer

The adsorption of lysozyme on mixed phosphatidyl choline-cardiolipin vesicles was studied at pH 4.0 and 6.0. The binding constants at both pH were determined at 0 and 22 degrees C. The presence of maximum on the adsorption isotherm at pH 6.0 was interpreted as an indication of the formation of two types of the protein-lipid complexes. This interpretation was confirmed by electron-microscopic observations. On the other hand, at pH 4.0 only one type of the protein-lipid complex was formed. The lysozyme conformation in solution and in the protein-lipid complexes was studied by circular dichroism. It was found that at acidic pH the lysozyme molecule contains a higher per cent of alpha-helix segments than at neutral pH. As follows from the measurements of lysozyme distribution in two phase systems the increase in alpha-helicity results in the formation of hydrophobic patches on the surface of the protein molecule. The results of the present work and of the previous studies of the interaction of red- and oxy- form of cytochrome C with phospholipid allow the conclusion that for peripheral proteins the nature of protein-lipid interactions is determined by the protein alpha-helix content and by hydrophobic pattern of the protein molecule surface.     

A mutant T4 lysozyme (Val 131----Ala) designed to increase thermostability by the reduction of strain within an alpha-helix

An attempt has been made to identify residues in T4 phage lysozyme that may have strained conformations and, by appropriate site-directed replacements, to reduce this strain and thus increase the thermostability of the protein. Valine 131, within alpha-helix 126-134, was identified as a potential candidate. Its side-chain rotational angle, chi 1, differs by approximately 18 degrees from the low-energy trans configuration. In addition, it is largely solvent exposed, yet is held in a rigid conformation. The mutant protein with Val 131 replaced by alanine was constructed and found to have a melting temperature 0.9 degrees C higher than that of wild-type lysozyme at pH 2.8. As a control, the mutant Val 131----Thr was also constructed and its melting temperature was found to be marginally lower than wild type. High-resolution crystal structure determinations of the mutant lysozymes show that their structures are virtually identical with that of wild-type lysozyme, except for the Val----Ala or Val----Thr replacement. Analysis of the different structures suggests that the design of the Val----Ala substitution was, in principle, successful, although the apparent gain in stability caused by reduction in strain is modest and is somewhat offset by the loss of hydrophobic interactions and by entropic effects. The results also help to provide a structural rationalization for the experimental and empirical observations that alanine has a higher helix propensity than valine or threonine.     

Cellular and humoral aspects of innate immunity in the shark

Survival of many species depends, to a great extent, on their innate immunity. Innate immunity in the nurse shark (Ginglymostoma cirratum), a primitive elasmobranch, has been shown to consist of components, both humoral and cellular, which are in some respects similar to those found in mammals and other vertebrates. Innate immune factors present in the shark include complement (a complex system of serum proteins) and antibacterial proteins and enzymes, such as lysozyme. Shark complement, although opsonic and lytic in nature, differs from classical mammalian complement in the number of functionally distinct components involved in the activation sequence. Functional and structural analogues of several mammalian complement proteins have been isolated from the shark, and activation of shark serum by lipopolysaccharide or zymosan produces anaphylatoxin-like ligand(s) inducing mammalian smooth muscle contraction and chemotaxis of human leucocytes in vitro. Lysozyme activity has been recovered from shark leucocyte lysates, which also contain antibacterial peptides, distinct from lysozyme. The composition and antibacterial activity of shark leucocyte granules, the putative source of the activity, is under investigation. Cellular aspects of the inflammatory response which is an integral component of innate immunity, are leucocyte phagocytosis and chemotaxis. Both processes are functions of two distinct shark cell types, the granulocyte and the monocyte-macrophage. It should be noted that the innate resilience of the nurse shark is also augmented by a large pool of serum natural antibodies, which can account for as much as 45% of the total serum protein.     

Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 A resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.     

Studies on the substrate specificity and inducibility of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system in Bacillus megaterium A.T.C.C. 13368 was investigated with regard to its appearance in the cell with respect to the growth curve of the organism, with regard to its inducibility by a number of agents (among them some of the classical inducers of the mammalian liver microsomal cytochrome P-450 system) and with regard to its capacity to convert non-steroidal substances into oxygenated compounds. The enzyme was found to reach a maximum concentration in the cell during the stationary phase of the growth curve. Of all the agents tested as inducers, none showed any capacity to induce cytochrome P-450meg. Finally, of the substances tested as substrates only aniline (p-hydroxylation) was metabolized by the microbial enzyme system. This conversion might be related to the general oxygenase activity of haemoproteins. It is concluded that the substrate specificity of the B. megaterium hydroxylase system is narrow.