Trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus: fine structure and immunohistochemical detection of elastin

Immunohistochemical and ultrastructural methods were used to examine the distribution of elastin and the fine structure of the trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus. The cells and matrix, as well as the overall organization of these components, in larval and adult trabecular cartilage resemble those of adult annular and piston cartilages (Wright and Youson: Am. J. Anat., 167:59-70, 1983) Chondrocytes are similar to those in hyaline cartilage. Lamprin fibrils and matrix granules, but no collagen fibrils, are found in a matrix arranged into pericellular, territorial, and interterritorial zones. Branchial, pericardial, and nasal cartilages differ from trabecular, annular, and piston cartilages in the organization of their matrix and in the structural components of their matrix and perichondria. Furthermore, immunoreactive elastin-like material is present within the perichondria and peripheral matrices of nasal, branchial, and pericardial cartilages in both larval and adult lampreys. Oxytalan, elaunin, and elastic-like fibers are dispersed between collagen fibers in the perichondrium. The matrix contains lamprin fibrils, matrix granules, and a band of amorphous material, which is reminiscent of elastin, in the periphery bordering the perichondrium. The presence of elastic-like fibers and elastin-like material within some lamprey cartilages implies that this protein may have evolved earlier in vertebrate history than has been previously suggested.     

Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.     

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials

Background

Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.

Methods

Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.

Results

MS² data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.

Conclusions

The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.

General significance

The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.     

Cartilage type IX collagen is cross-linked by hydroxypyridinium residues

Type IX collagen, a recently discovered, unusual protein of cartilage, has a segmented triple-helical structure containing interchain disulfides. Its polymeric form and function are unknown. When prepared by pepsin from bovine articular cartilage, type IX collagen was found to contain a high concentration of hydroxypyridinium cross-links, similar to that in type II collagen. Fluorescence spectroscopy located the hydroxylysyl pyridinoline and lysyl pyridinoline cross-linking residues exclusively in the high-molecular-weight collagen fraction, from which they were recovered predominantly in a single CNBr-derived peptide. The results point to a structural role for type IX collagen in cartilage matrix, possibly as an adhesion material to type II collagen fibrils.     

Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH₄. On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.     

The structure and mechanical properties of the proteins of lamprey cartilage

The cyanogen bromide‐resistant proteins of lamprey cartilage are biochemically related to the mammalian elastic protein, elastin. This study investigates their mechanical properties and enquires whether, like elastin, long‐range elasticity arises in them from a combination of entropic and hydrophobic mechanisms. Branchial and pericardial proteins resembled elastin mechanically, with elastic moduli of 0.13–0.35 MPa, breaking strains of 50%, and low hysteresis. Annular and piston proteins had higher elastic moduli (0.27–0.75 MPa) and larger hysteresis. Exchanging solvent water for trifluoroethanol increased the elastic moduli, whereas increasing temperature lowered the elastic moduli. Raman microspectrometry showed small differences in side‐chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were like those of elastin, preponderantly unordered, which probably confer the conformational flexibility necessary for entropy elasticity. Piston and annular proteins showed the strongest interactions with water, suggesting, together with the mechanical testing data, a greater role of hydrophobic interactions in their mechanics. Two‐photon imaging of intrinsic fluorescence and dye injection experiments showed that annular and piston proteins formed closed‐cell honeycomb structures, whereas the branchial and pericardial proteins formed open‐cell structures, which may account for the differences in mechanical properties. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 187–202, 2015.     

The unusual cartilaginous tissues of jawless craniates, cephalochordates and invertebrates

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.     

Molecular-level characterization of elastin-like constructs and human aortic elastin

This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS² data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin.     

Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.

The cell line, RCS-LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N-procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. Retention of the N-propeptide therefore does not appear to interfere with the interactions needed to form cross-links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross-linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen. The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N-propeptides.     

On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences.

The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.     

Effect of age on pyridinoline and pentosidine matrix cross-links in the desert sand rat intervertebral disc

Spondylosis in the desert sand rat (Psammomys obesus) has been studied as a model for intervertebral disc degeneration. Reducing sugars, which react with protein amino groups to form a diverse group of moieties with fluorescence and cross-linking properties, have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. This study was undertaken to determine the changes in two matrix cross-links of the intervertebral disc and to study their association with aging. Two types of cross-links were studied: the physiological cross-link, pyridinoline, which is initiated by lysyl oxidase; and the non-enzymatically initiated cross-link, pentosidine. A significant increase in pentosidine, but not pyridinoline, was observed in the intervertebral disc with aging. Radiological, histological and biochemical findings support a hypothesis that subchondral bone responses, marked by increased bone density, contribute to alterations in the intervertebral disc. Cross-link changes in the structural proteins of the disc may contribute to the progressive fibrocartilage degradation typical of intervertebral disc disease as an effect of age.     

A model two-component system for studying the architecture of elastin assembly in vitro

Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.     

Induced Collagen Cross-Links Enhance Cartilage Integration

Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2–2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.     

Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis.

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1.     

Histochemical identification of sulfation position in chondroitin sulfate in various cartilages

The ability of chondrocytes to synthesize chondroitin-4-sulfate (C4S) as opposed to chondroitin-6-sulfate (C6S) is a phylogenetically related phenomenon seen among adult higher vertebrates and developmentally during the embryogenesis of these vertebrates. While the embryonic cartilage may be initially a C6S matrix, C4S synthesis is seen to develop with time. We have histochemically localized these differences in sulfation with the cationic carbocyanine dye, Stains-all, in a spectrum of cartilages that vary in the sulfation position of their chondroitin sulfate. Cartilages from the rat and rabbit that are predominantly C4S stained magenta at pH 4.3, while the C6S-rich cartilage matrices from the regenerating rabbit ear and lamprey cranium stained blue. Embryonic chicken cartilages develop a gradient of magenta matrix with age, with increased concentration toward the articular surface. Both magenta and blue matrices were absent after pretreatment with chondroitinase ABC but were present after Streptomyces hyaluronidase digestion. The magenta staining was a property of the cartilage matrix as a whole, since isolated C4S and C6S stained blue. The differential staining was seen at pH 4.3, but not at pH 8.8, suggesting an interaction between the chondroitin sulfate and the adjacent tissue proteins.     

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.     

Decay of vertebrate characters in hagfish and lamprey (Cyclostomata) and the implications for the vertebrate fossil record

The timing and sequence of events underlying the origin and early evolution of vertebrates remains poorly understood. The palaeontological evidence should shed light on these issues, but difficulties in interpretation of the non-biomineralized fossil record make this problematic. Here we present an experimental analysis of decay of vertebrate characters based on the extant jawless vertebrates (Lampetra and Myxine). This provides a framework for the interpretation of the anatomy of soft-bodied fossil vertebrates and putative cyclostomes, and a context for reading the fossil record of non-biomineralized vertebrate characters. Decay results in transformation and non-random loss of characters. In both lamprey and hagfish, different types of cartilage decay at different rates, resulting in taphonomic bias towards loss of 'soft' cartilages containing vertebrate-specific Col2α1 extracellular matrix proteins; phylogenetically informative soft-tissue characters decay before more plesiomorphic characters. As such, synapomorphic decay bias, previously recognized in early chordates, is more pervasive, and needs to be taken into account when interpreting the anatomy of any non-biomineralized fossil vertebrate, such as Haikouichthys, Mayomyzon and Hardistiella.     

Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures

Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis. Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin. The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration. Primary hamster lung cell cultures were utilized. Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone. Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine. These cross-links are found only in elastin. Addition of [14C] lysine to cultures results in uptake of the radiolabel into the cross-links. Cross-links were isolated and identified using chromatography and electrophoresis. At all doses of amiodarone, elastin synthesis was seen to increase above control levels. Light and electron microscopy confirmed the presence of an extracellular matrix. The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.     

Structural determinants of cross-linking and hydrophobic domains for self-assembly of elastin-like polypeptides

Elastin is a major structural protein found in large blood vessels, lung, ligaments, and skin, imparting the physical properties of extensibility and elastic recoil to these tissues. To achieve the required structural durability of the elastic matrix, the elastin monomer, tropoelastin, undergoes ordered assembly into a covalently cross-linked, fibrillar polymeric structure. Human tropoelastin consists of 34 exons coding for alternating hydrophobic and cross-linking domains. Using a series of well-defined recombinant polypeptides based on human elastin sequences mimicking native elastin, we have previously investigated the role of sequence and context of hydrophobic domains in elastin self-assembly. Here, we demonstrate that the structure of both cross-linking and hydrophobic domains have significant effects on the assembly of these polypeptides. Removing a putative flexible hinge region in the center of a cross-linking domain substantially increased the alpha-helical content and strongly promoted their self-aggregation. However, while trifluoroethanol (TFE) promoted and urea inhibited self-assembly of these polypeptides, these effects were not predominantly due to altered alpha-helicity of the polypeptides. Our results suggest that, while increased alpha helicity also favors this process, the major effect of TFE to promote organized self-assembly of elastin-like polypeptides is likely related to direct effects of this cosolvent on hydrophobic domains. Such simple elastin polypeptide models can provide an important tool for understanding the relationships between sequence, structure, and polymeric assembly of elastin.