Sulfation as a collagen-specific reaction The ultrastructure of sulfate collagen,basement membranes and reticulin fibers as shown by topo-optical staining reactions

Summary Sulfation induces hyperbasophilia in connective tissue structures (fibrillary collagen, basement membranes and reticulin fibers), which appear metachromatic with toluidine blue at pH 1.0 and strongly birefringent with inversion of their positive birefringence into negative birefringence indicating transversally oriented and closely packed dye molecules on the micellar surface of collagen. Quantitative studies of the sulfation induced topooptical staining reaction following blocking of the vicinal glycol groups by periodate and the enzymatic removal of AMP support the view that carbohydrate glycol groups play only a minor part and the OH side-groups of the collagen peptide chains play the major part in the sulfation reaction of fibrillary collagen and basement membranes.After blocking of the vicinal glycol groups of carbohydrate components by periodate, sulfation induced toluidine blue hyperbasophilia with strong negative birefringence associated with selective proteolytic sensitivity are collagen-specific characeteristics due to sulfate esterification on the OH groups of the peptide chains of collagen, which provide new approach to the study of the ultrastructure of connective tissue elements in physiology and pathology.     

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.     

Thermotropic behavior of glycosphingolipids in aqueous dispersions

The thermotropic behavior of 20 chemically related glycosphingolipids (GSLs) of high purity, containing neutral and anionic carbohydrate residues in their oligosaccharide chains, was studied by high-sensitivity differential scanning calorimetry. In general, the polar head group of GSLs appears to be one of the major determinants of their phase behavior. Compared to phospholipids, the presence of the carbohydrate rather than the phosphorylcholine moiety in the polar head group and a sphingosine base in the hydrocarbon portion of GSLs reduces the effect on the transition temperature (Tm) brought about by increasing the number of methylene groups in the amide-linked fatty acyl chains. For simple neutral GSLs, the Tm's were 20-40 degrees C higher than those of phospholipids with comparable hydrocarbon chains. As the oligosaccharide chain of GSLs becomes more complex, the excess heat capacity, Tm, enthalpy (delta Hcal), and entropy of the transition decrease proportionally to the number of carbohydrate residues present in the polar head group. The Tm and delta Hcal for anionic GSLs were 16-25 degrees C and 1-3 kcal mol-1 lower than those of neutral GSLs with comparable oligosaccharide chains. A linear dependence of delta Hcal with Tm was found. However, the slopes of these plots were different for neutral and for anionic GSLs, suggesting different types of intermolecular organizations for the two. The Tm and delta Hcal were linearly dependent on the molecular area of both neutral and anionic GSLs; this indicated that the influence of the complexity of the polar head group in GSLs for establishing the thermodynamic behavior may be mediated by the intermolecular spacings.     

Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides

Carbohydrate-protein linkages of three types are found in the connective tissue proteoglycans; these linkages involve the following monosaccharide-amino acid pairs: xylose-serine; N-acetylglucosamine-asparagine; and N-acetylgalactosamine-threonine (or serine). The biosynthesis of carbohydrate groups containing linkages of the latter two types presumably occurs by the same pathways that have been well established for many glycoproteins, but details of these processes as they pertain to proteoglycans are not yet known. Initiation of polysaccharide chains linked by the xylose-serine linkage takes place by direct transfer of xylose from UDP-xylose to the hydroxyl groups of specific serine residues in the core proteins of the respective proteoglycans, and the xylosyltransferase catalyzing these reactions has been detected in the rough endoplasmic reticulum of embryonic chick chondrocytes. Although the completed or nascent core proteins are the natural substrates for xylose transfer in the intracellular assembly of proteoglycans, a survey of potential exogenous substrates has shown that small peptides containing alternating serine and glycine residues may also serve as acceptors in this reaction. Nevertheless, larger substrates are preferred, such as chondroitin sulfate proteoglycan, which has been deglycosylated by Smith degradation or HF treatment, or silk fibroin, which contains Ser-Gly pairs. In contrast to the sulfated polysaccharides, which are synthesized by carbohydrate transfer to protein in the endoplasmic reticulum and the Golgi apparatus, hyaluronic acid is formed in the plasma membrane by a different mechanism. The reaction by which chains are initiated is not yet known, but recent work by Prehm suggests that this process occurs either by transfer of the glucuronosyl component of UDP-glucuronic acid to UDP-N-acetylglucosamine or by the converse reaction, i.e., transfer of the N-acetylglucosaminyl unit of UDP-N-acetylglucosamine to UDP-glucuronic acid.     

Biochemical and physiochemical characterization of pepsin-solubilized type-II collagen from bovine articular cartilage.

Solubilization of collagen from bovine articular with pepsin requires the preliminary extraction of proteoglycans from the ground substance. Biochemical and physiochemical properties of this pepsin-solubilized collagen are independent of the pretreatment (extraction with 1.5M-CaCl2, 5M-guanidinium chloride or 0.2M-NaOH) and of the age range (2-4-year-old and 2-month-old animals). Characterization of the de-natured components, of the CNBr peptides and of the amino acid and cross-link composition shows that the collagen of the hyaline cartilage is all type II. Electrical birefringence measurements showed the presence of tropocollagen molecules (length 280nm) and molecules whose length is slightly less than twice that of the tropocollagen molecules. This latter molecule may be a dimer composed of two monomers linked by intermolecular head-to-tail bonds and whose theoretical length (530nm), according to the quarter-stagger theory, is in good agreement with our measured values (510-530nm). We have verified that the beta-components of this collagen are formed of two alpha-chains linked by the stable intermolecular bond, dehydrodihydroxylysinonorleucine. These dimeric molecules are absent from solutions of skin collagen whose beta-components possess only aldol-type intramolecular cross-links. Although reconstituted fibres from solutions of skin and cartilage collagen are similar, the segment-long spacing crystallites formed with pepsin-solubilized cartilage collagen present a symmetrical and dimeric form corresponding to the lateral aggregation of two monomers with an overlap (90nm) of the C-terminal ends.     

Different levels of glycosylation contribute to the heterogeneity of alpha 1(II) collagen chains derived from a transplantable rat chondrosarcoma

Three collagen fractions, each of which contain molecules composed of alpha 1(II) chains, have been isolated from pepsin-solubilized rat chondrosarcoma collagen. One fraction could be selectively precipitated from the pepsin digest at 0.7 M NaCl. Two additional fractions were obtained on chromatography of the collagen precipitating at 1.2 M NaCl on carboxymethyl cellulose under nondenaturing conditions. When chromatographed on carboxymethyl cellulose under denaturing conditions, each fraction contained components eluting in the position expected for alpha 1(II) chains. One of the fractions precipitating at 1.2 M NaCl contained the recently described 1 alpha and 2 alpha chains in addition to material eluting as alpha 1(II) chains. Comparison of the chains eluting as alpha 1(II) chains in the various fractions with respect to amino acid composition, carbohydrate content, and cyanogen bromide-cleavage products showed that they differed only in the number of glycosylated hydroxylysyl residues. In this regard, alpha 1(II) chains obtained from collagens precipitated at 1.2 M NaCl exhibited significantly higher levels of glucosylgalactosylhydroxylysyl residues than alpha 1(II) chains precipitated at 0.7 M NaCl. These results indicate that molecules composed of alpha 1(II) chains are heterogeneous with respect to levels of hydroxylysine-linked carbohydrate moieties and that the more highly glycosylated molecules require higher salt concentrations for precipitation from acidic solutions. The data also indicate that a proportion of the more highly glycosylated alpha 1(II) chains are involved in the formation of one or more molecular species with 1 alpha and 2 alpha chains.     

Effect of Ca2+ on conformation of actin in the F-actin--heavy meromyosin complex

The polarized fluorescence of intrinsic tryptophan residues and the birefringence of ghost muscle fibres of rabbit were measured during thin filaments binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains and to those devoid of them with a view of investigating conformational changes in F-actin. Ca2+ binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains was shown to affect the character of these changes during the formation of the F-actin - heavy meromyosin complex.     

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.     

Undecagold labeling of a glycoprotein: STEM visualization of an undecagoldphosphine cluster labeling the carbohydrate sites of human haptoglobin-hemoglobin complex

A new type of label for electron microscopy has been introduced recently which consists of 11 gold atoms in a compact stable cluster with an organic shell composed of primary amine-substituted phosphine ligands. The radius of the cluster is about 10 A. The (phosphine ligand) amines can be derivatized or allowed to react directly forming covalent bonds to specific sites of other molecules. This report describes the specific labeling of carbohydrate moietis on the glycoprotein human haptoglobin (Hp) in the haptoglobin-hemoglobin complex (Hp X Hb). The Hp X Hb complex is easily recognized in the EM as a barbell-shaped molecule. Only the Hp portion contains carbohydrate (eight carbohydrate chains per Hp X Hb). The carbohydrate moieties of the Hp X Hb complex were oxidized by sodium periodate to produce aldehydes. The primary amines on the undecagold cluster were allowed to react with the aldehyde residues to produce Schiff's base linkages which were subsequently reduced with sodium borohydride. Micrographs obtained on the Brookhaven National Laboratory high-resolution scanning transmission electron microscope (STEM) showed the undecagold label to be localized in a region known to be occupied by the heavy chains of haptoglobin. The amount of labeling was found to be two to four gold clusters per molecule when excess label was reacted. The variation in position of the label is discussed and may be due to flexibility of the carbohydrate chains. Control experiments ruled out nonspecific binding of the gold cluster to the Hp X Hb. The high chemical specificity of the reaction and the high resolution of the gold cluster should make this new label of widespread value in studies of other glycoproteins or carbohydrate-bearing molecules.     

A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin).

We have determined the primary structure of a 59 kd collagen binding protein which is present in many types of connective tissues, e.g. cartilage, tendon, skin, sclera and cornea. The amino acid sequence, deducted from a 2662 bp cDNA clone, predicts a 42 kd protein with a high content of leucine residues. Most of the protein consists of homologous 23 amino acid residues repeats with predominantly leucine residues in conserved positions. Similar leucine rich repeats have been identified in a number of proteins including the small interstitial proteoglycans decorin and PG-S1. The 59 kd protein and the two proteoglycans are homologous in their entire sequences suggesting that they have evolved from a common ancestral gene. The 59 kd protein and decorin are also functionally related in that both bind to collagen type I and II, and affect their fibrillogenesis. The substitution with glycosaminoglycan chains appears to be a feature shared by all three members of this family of leucine rich motif extracellular proteins, since the 59 kd protein isolated from cartilage is substituted with at least one keratan sulfate chain.     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.     

A quantitative collagen fibers orientation assessment using birefringence measurements: calibration and application to human osteons

Even though mechanical properties depend strongly on the arrangement of collagen fibers in mineralized tissues, it is not yet well resolved. Only a few semi-quantitative evaluations of the fiber arrangement in bone, like spectroscopic techniques or circularly polarized light microscopy methods are available. In this study the out-of-plane collagen arrangement angle was calibrated to the linear birefringence of a longitudinally fibered mineralized turkey leg tendon cut at variety of angles to the main axis. The calibration curve was applied to human cortical bone osteons to quantify the out-of-plane collagen fibers arrangement. The proposed calibration curve is normalized to sample thickness and wavelength of the probing light to enable a universally applicable quantitative assessment. This approach may improve our understanding of the fibrillar structure of bone and its implications on mechanical properties.     

Distribution of receptor sites on large glycoprotein molecules by electron microscopy. Application to epiglycanin.

Electron microscopy was used to map the loci of immunochemically active sites on individual glycoprotein molecules. The positions of specific galactose residues and asparagine-linked carbohydrate chains containing specific mannose residues in epiglycanin, a glycoprotein of extended conformation from the surface of TA3 mouse mammary tumour cells, were observed in complexes with Ricinus communis toxin and concanavalin A respectively. The maximum number of Ricinus communis toxin molecules attached to a single epiglycanin molecule was 23, and the average number was 16. Only one concanavalin A molecule was observed attached to any epiglycanin molecule, and this at one end of the molecule, suggesting the presence of only one receptor for this lectin. By means of this new approach for mapping specific residues, evidence has been obtained that suggests microheterogeneity in epiglycanin with respect to the locations of carbohydrate chains containing receptors for Ricinus communis toxin.     

Axial ranking of residues in collagen in relation to edge association and fibril formation

In our earlier analysis of intermolecular interactions between collagen molecules, a major concern with the program employed is that it compared numbers of interactions between residues located on edges of defined, identical width and thus would not necessarily compare the same number of residues in each edge. This would be particularly true of some values of θ where well-defined vertical ranking of residues occurs. We have examined ranking of residues in relation to intermolecular edge association between bovine skin [α1(I)]₃ model collagen molecules by utilizing two different methods of counting intermolecular interactions between residues. The interaction peaks at θ = 27.69° and 36.00° are absent or relatively less intense in the plots obtained by utilizing radial distances between interacting residues instead of vertical bands of defined width. These studies suggest caution in accepting recently reported analyses of superhelix coiling of the collagen molecule which point to values of 27.69° or 36.00° for the twist of the superhelix. Although intramolecular interactions clearly point to interaction of collagen molecules at D intervals, they are insufficiently restricted in distribution to provide a reliable estimate of the superhelix angle by procedures so far employed.     

Identification of the depth of burn injury by collagen stainability

Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old. The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls. The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls. The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.     

Composition and structure of the pericellular environment. Physiological function and chemical composition of pericellular proteoglycan (an evolutionary view).

Connective tissue cells exist in a meshwork of insoluble fibres, the interstices of which are filled with soluble, high molecular mass, anionic material of a predominantly carbohydrate nature. The interactions of fibres with the interfibrillar material are central to the discussion of connective tissue physiology. As with all soluble polymers, the interfibrillar polyanion tends to "swell' and the tangled mass of chains offers considerable resistance to penetration by the large insoluble fibres. The consequent pressure to "inflate' the fibrous network is important in giving elasticity to cartilage, transparency to cornea, etc. Branched structures (of proteoglycans) and straight-chain forms (of hyaluronate) are compared for their ability to fulfil these functions. Apart from their physical ("non-specific') roles proteoglycans and glycosaminoglycans are able to interact physicochemically with, for example, collagen in ways which show considerable specificity, and which presumably are important in the laying down of the fibrous network as well as in maintaining its mechanical integrity. It is proposed that the role played by radiation, particularly as mediated via the hydrated electron (eaq) was dominant in the pre- and post-biotic evolution of pericellular environments.     

Carbohydrate content of bovine collagen preparations

Collagen preparations from bovine tissues were analysed for their carbohydrate content. Crude preparations of tropocollagen and polymeric collagen were found to be contaminated with considerable amounts of mannose, fucose and hexosamine, sugars known to be present in the mucoprotein of the interfibrillar material with which collagen is associated in vivo. A pure preparation of tropocollagen obtained by ethanol precipitation procedures contained only galactose and glucose in the approximate ratio of 7:3 residues/3000 amino acid residues. Purification of crude polymeric collagen by EDTA extraction or by crude bacterial amylase extraction considerably decreased the mucoprotein contamination, particularly in the enzymic treatment, which yielded a preparation containing predominantly galactose and glucose in the ratio of 4:2 residues/3000 amino acid residues. The results confirm previous work that demonstrated the purity of these collagen preparations as inferred by amino acid analysis. The results also indicate the suitability of the pure tropocollagen and the amylase-extracted polymeric collagen for studies on the role of the carbohydrate residues in intramolecular and intermolecular cross-linking in collagen.     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

On the role of the collagen carbohydrate residues in the platelet: Collagen interaction

It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To rest this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantlly reduced its ability to interact with platelets. The extent of inhibition paralled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.