Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.

The amino acid sequence coded by the rarely expressed exon 26A of human elastin contains a stable beta-turn with chemotactic activity for monocytes.

The structural and biological properties of the amino acid sequence coded by the rarely expressed exon 26A of human elastin were investigated. The C-terminal portion of this sequence, corresponding to residues 600-619 of human tropoelastin, REGDPSSSQHLPSTPSSPRV and three shorter derived peptides, LREGDPSS, SSSQHLPS, and LPSTPSSP, were synthesized and studied. Spectroscopic analyses by CD and NMR have identified a type II beta-turn within the sequence REGD of the octapeptide LREGDPSS. This structural motif was found also in the tetrapeptide REGD in both trifluoroethanol and water. The CD spectrum of the tetrapeptide REGD in trifluoroethanol was consistent with a pure type II beta-turn. A high chemotactic activity for monocytes was exhibited by the structured peptides REGD (CI 0.90 at 10(-)7 M) and LREGDPSS (CI 0.80 at 10(-)11 M), at variance with the unfolded peptides LPSTPSSP and SSSQHLPS, suggesting that this activity is strictly correlated with folded structures. Because the exon 26A of human elastin is expressed in the neointima of hypertensive pulmonary arteries, and macrophages are present in this pathologic tissue [Liptay et al. (1993) J. Clin. Invest. 91, 588-594], the chemotactic activity for human monocytes reported in this paper is consistent with an active role played by the exon 26A in inducing the migration of the monocyte/macrophage cells to the neointima.

Immunolocalization of a 35K structural glycoprotein to elastin-associated microfibrils.

In this study, a rabbit antiserum directed against a 35K glycoprotein (35K-GP), extracted from connective tissue, was used to examine the localisation of this antigen within foetal bovine ligamentum nuchae at stages of development preceding (4th month) and coinciding with (7th month) active elastin biosynthesis. In these tissues the antibody, detected by a colloidal gold conjugate technique, localised specifically to 11-nm fibrils, identified as the microfibrillar component, present both in the form of independent bundles and in association with elastin in the developing elastic fibres. No other connective tissue component was recognised by the antibodies which had been purified by affinity chromatography. The ability of the antibodies to bind to the microfibrils appeared to be dependent on embedding in LR White resin, as colloidal gold binding was greatly reduced in tissue embedded in epoxy resin. These results are discussed with respect to the role that 35K-GP may play in the morphogenesis of the elastic fibre.

Morphogenesis of the elastic fiber: an immunoelectronmicroscopy investigation.

In this study a rabbit antiserum against human aortic elastin, which showed a high degree of species specificity in ELISA tests, was used to examine elastin fiber formation in the human fetal aorta between the ages of 14 and 23 weeks. Elastin was first detected by the antibody in the matrix of the 14-week-old specimen in association with the microfibrillar component. At this stage of development, the sections did not reveal structures morphologically identifiable as elastin. By the 17th week, discrete loci of elastin deposition were observed together with well-defined elastin fibrils. Only by the 23rd week did the aorta show the characteristic layering of elastic fibrils separating the myoblasts of the tunica media. In the latter specimen, the newly synthesized uncrosslinked elastin appeared to be unevenly distributed on the surface of elastin fibrils where it formed continuous strips of variable width arranged mostly in the form of spirals. This observation is discussed with respect to the proposals that the morphogenesis of elastic tissue is a dynamic process involving a close interrelationship between elastic fibrils and elastogenic cells and the morphogenetic movement of elastogenic cells plays an important role not only in the growth of elastic fibrils but also in the ultrastructural organization of the tissue.

Age-related changes in composition and mechanical properties of the tunica media of the upper thoracic human aorta.

A cylindrical segment, free of complex atherosclerotic lesions, was resected at autopsy from each of 59 descending human thoracic aortas by cutting just below the level of the first pair of intercostal arteries and 35 mm distal to this incision. Each isolated tunica media was defatted and subjected to successive treatment with EDTA-Tris, 5 M guanidine hydrochloride-Tris, 5 M guanidine hydrochloride-Tris-DTE, collagenase and either trypsin or hot alkali. After each extraction or digestion, the dimensions and weight of the segments were measured and the extracted materials were analyzed and quantitated. This allowed the total content of the various components of the tunica media to be assessed by both gravimetric and analytical means. An age-related rise was observed in the total content of the following components: proteins and glycoproteins soluble in chaotropic solvents (ranging from 24 mg/cm in the youngest samples to 46 mg/cm in the oldest) and collagen (38 mg/cm to 69 mg/cm). In contrast, the total content of elastin remained constant at 70 mg/cm at all ages, but its concentration decreased due to the rise in the concentration of the other tissue components as the tunica media thickened with age. It was also noted that with increasing age there was an accumulation of protein(s) which could not be solubilized by extraction with chaotropic agents or with collagenase, but which could be removed by treatment with either trypsin or hot alkali. Mechanical measurements conducted before and after trypsin digestion on samples previously subjected to purification with the first four agents used suggest that this accumulated protein(s) influenced the elastic response of the tissue to the applied stress by increasing the incremental modulus, the breaking stress, and the hysteresis. After the removal of this additional protein(s), the mechanical behavior of the elastin component was found to be identical in all samples, irrespective of age. It is therefore proposed that the morphological changes and the stiffening observed in the aging aortic wall are not due to degradation of its elastin network but to variations in the supramolecular organization of connective tissue components.

Characterization of a structural glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity.

A structural glycoprotein has been extracted from bovine ligamentum nuchae by using 5 M guanidine hydrochloride containing a disulfide bond reducing agent and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse, with a molecular weight of approximately 34000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetylglucosamine, galactose, and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity by using [3H]lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2-h incubation was approximately 60 X 10(4) dpm/mg of purified protein. Addition of 10 mM lysine resulted in an approximately 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using [3H]lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27 mM beta-aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by approximately 14%. In the absence of 5 M guanidine hydrochloride and reducing agent, the glycoprotein undergoes aggregation which in the presences of copper ions results in the formation of cylindrical tactoids, the diameter of which (11 nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibers.

Characterization of a self-associating glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity.

A homogeneous preparation of glycoprotein (apparent molecular weight 34,000) has been isolated from bovine ligamentum nuchae by treatment of the tissue with 5 M guanidine HCl and 2-mercaptoethanol. The preparation exhibits amine oxidase activity which is only marginally inhibited by beta-aminopropionitrile.

The ultrastructure and mechanics of elastic ligaments.

The major central part of elastic ligaments is constituted by longitudinally orientated elastic fibres, each surrounded by a sheath of spirally orientated collagen fibrils with an intervening thin layer of microfibrils. In the terminal region of such ligaments the orientation of the collagen fibrils is longitudinal and the elastic fibres end within the ligament without any attachment to bone. The ligament response to a first, unphysiologically high load produces a continuous stress-strain curve. On the other hand the curve becomes biphasic after repeated application of such loads. It is suggested that three factors are involved in this mechanical response, namely, frictional forces between elastic fibres and their sheaths, compressive forces exerted upon elastic fibres by their sheaths, and intre-sheath friction due to rearrangement of the constituent collagen fibrils.

X-ray analysis of enzymically purified elastin from bovine ligamentum nuchae.

Insoluble elastin has been isolated from bovine ligamentum nuchae by treatment with quanidine and dithiothreitol followed by digestion with collagenase, purified by affinity chromatography. The preparation was subjected to both wide- and low-angle X-ray analysis. The wide-angle diffraction patterns of relaxed and stretched specimens showed only two broad diffraction rings, corresponding to spacings of 4.5 and 9.3 A. No significant reflections were visible in the low-angle diffraction pattern of unstretched specimens, but on stretching an equatorial reflection was produced, corresponding to spacings of between 45 and 50 A.