Barbara Faris

Identification of a new crosslinking amino acid in elastin

Abstract Recently, Partridge et al . (1963) described the occurrence in elastin of two hitherto unrecognized amino acids for which evidence has now accrued to show their origin from lysine residues in a “pre-elastin” (Partridge et al ., 1964; Miller et al ., 1964). These substances, called desmosine and isodesmosine, are in fact quaternary pyridinium compounds with four side-chain substituents that function in crosslinking of elastin chains (Thomas et al ., 1964). An examination of peptide fractions of elastin selected for enrichment with respect to the desmosines revealed the presence of an additional amino acid, called X 4′ also previously undescribed (Franzblau et al ., 1965). The present communication presents data to show that X 4 can be represented by the formula, N ϵ -(5-amino 5-carboxypentanyl)-lysine.

The synthesis of connective tissue protein in smooth muscle cells

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

Aging effects on the elastin composition in the extracellular matrix of cultured rat aortic smooth muscle cells.

SummaryElastin accumulation in the extracellular matrix of cultured rat aortic smooth muscle cells was monitored as a function of age. The effect of the animal donor age and time in culture in single or consecutive passages on the cells’ ability to accumulate total protein as well as elastin was evaluated. Smooth muscle cells were obtained from animals ranging in age from 2 d to 36 mo. Protein accumulation by the cells based on DNA content was similar regardless of which of the above aging parameters was examined. Although there were significant amounts of elastin present in the extracellular matrix of those cells originating from the younger animals (2 d and 6 wk old), little or none was detected in cell cultures derived from the oldest animals. A soluble elastin-like fraction which was isolated from the cultures of the 2-d-old rats seemed to be lacking in the cultures of cells from the 36-mo-old animals. This observation may, in part, explain the absence of insoluble elastin in the matrix of some cultures obtained from older animals. The data strongly suggest that the age of the donor animal from which the cells originate has the greatest influence on in vitro elastin accumulation.

The interaction of collagen with α1-acid glycoprotein

Abstract The influence of α 1 - acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the α 1 - acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45–55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and e-hydroxynorleucine.

Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts

The mechanism by which Clostridium difficile toxin B causes cells in culture to round was investigated. Cultured human lung fibroblasts and rabbit aortic smooth muscle cells were treated with partially purified or purified toxin B and monitored by light and transmission electron microscopy (TEM). Both preparations caused progressive cell rounding which correlated with disorganization of actin-containing myofilament bundles. Thin myofilaments became fragmented and finally disappeared (after 24 h) and dense bodies became more prominent, while all other organelles appeared unaffected.

Aldehydes in native and denatured calf skin tropocollagen

Some of the aldehydes which have recently been found in collagen and elastin, have been postulated to play a major role in the cross-linking and maturation of these two fibrous proteins. The aldehydes in elastin are derived from lysine residues in the protein backbone. By a presumed oxidation of the C-amino group, lysine is converted to a-amino adipic acid semialdehyde (Lent and Franzblau, 1968), an intermediate, which can either form a Schiff base with another lysine yielding dehydrolysinonorleucine or condense with another residue of the semialdehyde to yield the aldol product, c+amino adipic acid semialdehyde aldol (formula below), The presence of these compounds was recently demonstrated in elastin by Lent and Franzblau (Lent and Franzblau, 1968; Franzblau et al. 1965). Dehydrolysinonorleucine and a-amino adipic acid semialdehyde aldol may either serve as intermediates in the biosynthesis of desmosines or other as yet unknown cross-linking compounds, or may themselves serve the ultimate cross-linking role. In collagen, in addition to lysine, several other amino acids serve as aldehyde precursors. Since a-amino aldehydes derived from glycine, alanine,

Effect of varying amounts of ascorbate on collagen, elastin and lysyl oxidase synthesis in aortic smooth muscle cell cultures

In the presence of ascorbate, there is an increase in collagen synthesis with a concomitant decrease in insoluble elastin and lysyl oxidase activity in cultured rabbit aortic smooth muscle cells. While the addition of 0.5 micrograms ascorbate per ml of medium enhances collagen synthesis and accumulation, detectable insoluble elastin and lysyl oxidase activity remain essentially unchanged. However, at 2 micrograms ascorbate per ml, the integrity of the insoluble elastin is lost and lysyl oxidase activity is decreased. These studies suggest that by modifying the levels of ascorbate in the culture medium one can alter the nature of the extracellular matrix produced by the smooth muscle cells.

Lysine-derived cross-links in the egg shell membrane

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB3H4 reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells.

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.