The formation of hydroxyproline in collagen by cells grown in the absence of serum

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggest that reductant other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)(79), which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)(n) sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 degrees C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 degrees C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.     

Stabilization of collagen fibrils by hydroxyproline

The substitution of hydroxyproline for proline in position Y of the repeating Gly-X-Y tripeptide sequence of collagen-like poly(tripeptide)s (i.e., in the position in which Hyp occurs naturally) is predicted to enhance the stability of aggregates of triple helices, while the substitution of Hyp in position X (where no Hyp occurs naturally) is predicted to decrease the stability of aggregates. Earlier conformational energy computations have indicated that two triple helices composed of poly(Gly-Pro-Pro) polypeptide chains pack preferentially with a nearly parallel orientation of the helix axes [Nemethy, G., & Scheraga, H.A. (1984) Biopolymers 23, 2781-2799]. Conformational energy computations reported here indicate that the same packing arrangement is preferred for the packing of two poly(Gly-Pro-Hyp) triple helices. The OH groups of the Hyp residues can be accommodated in the space between the two packed triple helices without any steric hindrance. They actually contribute about 1.9 kcal/mol per Gly-Pro-Hyp tripeptide to the packing energy, as a result of the formation of weak hydrogen bonds and other favorable noncovalent interatomic interactions. On the other hand, the substitution of Hyp in position X weakens the packing by about 1.7 kcal/mol per Gly-Hyp-Pro tripeptide. Numerous published experimental studies have established that Hyp in position Y stabilizes an isolated triple helix relative to dissociated random coils, while Hyp in position X has the opposite effect. We propose that Hyp in position Y also enhances the stability of the assembly of collagen into microfibrils while, in position X, it decreases this stability.     

胶原海绵的羟脯氨酸含量测定

选用普通实验室均能实现的 Woessner第 法对自制胶原海绵和 Gelfix(国外样品 )进行了羟脯氨酸含量测定 ,结果表明 ,该方法操作简单 ,重复性好 :自制胶原海绵的羟脯氨酸含量稳定 ,与国外样品的羟脯氨酸含量接近 ,且与胶原蛋白的羟脯氨酸含量接近 ,证实了两种胶原海绵的纯度均较高     

Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro

Cloned, large vessel endothelial cells derived from fetal bovine and bovine calf aortas formed three-dimensional structures in vitro without tumor-conditioned medium or special substrata. Transmission electron microscopy showed the structures to be hollow tubes composed of typical endothelial cells with overlapping and interdigitating cytoplasmic processes typical of those seen in in vivo capillaries. The putative lumen of these tubes generally contained abundant electron-dense fibrous material, which by ruthenium red and indirect immunofluorescent staining appeared to be extracellular matrix. This suggests that the endothelial cell orientation in the tubes is the reverse of that normally found in in vivo vessels.     

The role of hydroxyproline in collagen folding: conformational energy calculations on oligopeptides containing proline and hydroxyproline

We have observed that the rate of folding of the enzymatically hydroxylated form of poly(Gly-Pro-Pro) into the triple-helical conformation is considerably higher than that of the unhydroxylated polypeptide [R. K. Chopra and V. S. Ananthanarayanan (1982) Proc. Natl. Acad. Sci. USA 79 , 7180–7184]. In this study, we examine a plausible kinetic pathway for triple-helix formation by selecting peptide models for the unhydroxylated collagen molecule, and computing their conformational energies before and after proline hydroxylation. Starting with the available data on the preferred conformations of proline- and hydroxyproline-containing peptide sequences, energy minimization was carried out on the following pairs of peptides: Gly-Ala-Pro-Gly-Ala and Gly-Ala-Hyp-Gly-Ala; Gly-Pro-Pro-Gly-Ala and Gly-Pro-Hyp-Gly-Ala; Gly-Ala-Pro-Gly-Ala-Pro and Gly-Ala-Hyp-Gly-Ala-Hyp. It was found that, with each pair of peptides, the energetically most favorable conformation (I) has an extended structure at the Gly-Ala or Gly-Pro segment and a β-bend at the Pro-Gly or Hyp-Gly segment. In the Hyp-containing peptides, this conformation is further stabilized by a (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond. Conformation I is lower in energy by about 6–13 kcal/mol of the peptide than the fully extended conformations that resemble the single collagen polypeptide chain and contain no intramolecular hydrogen bond. In contrast to the proline counterpart, the hydroxyproline-containing peptides are found capable of adopting a partially extended conformation that does not contain the β-bend but retains the (Hyp)OH…OC(Gly) hydrogen bond. The energy of this conformation is intermediate between conformation I and the fully extended conformation. The continuation of the β-bend along the chain is restricted by stereochemical constraints that are more severe in the latter two pairs of peptides than in the first pair. Such a restriction may be considered to trigger the “unbending” of the minimum energy conformation leading to its straightening into the fully extended conformation; the latter, in turn, would lead to triple-helix formation through favorable interchain interactions. We propose that the partially extended conformation in the Hyp-containing peptides could serve as a kinetic intermediate on the way to forming the fully extended conformation. Because of the (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond, this conformation would also serve to lock the trans geometry at the Gly-Ala(Pro) and Ala(Pro)-Hyp peptide bonds, thereby enhancing the rate of their helix formation. A scheme for collagen folding in proposed on the basis of these results.     

The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen

Protocollagen, a non-hydroxylated form of collagen, was extracted with cold 0.1 N acetic acid from embryonic tendon cells incubated with α,α′-dipyridyl and the protein was purified by controlled proteolytic digestion. The resulting modified protocollagen was shown to consist of polypeptides the same size as α1 and α2 chains of collagen and had a thermal transition by optical rotation similar to collagen. The T_m however was 24°, a value which was 15° lower than the T_m of an hydroxylated form of collagen from the same source. The results suggest that hydroxylated proline increases the thermal stability of collagen.     

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.     

Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells

The activities of four intracellular enzymes of collagen biosynthesis were assayed in freshly isolated rat peritoneal macrophages and mast cells and compared with the same enzymes in freshly isolated chick-embryo tendon cells. The macrophages were found to contain activities of all four enzymes, those of prolyl and lysyl hydroxylase being 7 and 12% respectively of those in the tendon cells when expressed per cell or 3 and 4% when expressed per unit of soluble cell protein. The corresponding values for hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase activities were about 82 and 68% or 32 and 24% respectively. When the macrophages were incubated in suspension with [(14)C]proline, they synthesized a small but significant amount of non-diffusible hydroxy[(14)C]proline. The synthesis per cell was only about 0.1% of that formed by the tendon cells, and its distribution between the cells and the medium also differed from that in the tendon cells. The hydroxy[(14)C]proline synthesized by the macrophages may be present in the Clq subcomponent of the complement, but its amount was too small to allow any characterization of the protein. All four enzyme activities, and in particular the two hydroxylysyl glycosyltransferase activities, seem to be present in macrophages in a large excess compared with the very low rate of synthesis of hydroxy-proline-containing polypeptide chains. The mast cell extract was found to inhibit all four enzyme activities, but even when corrected for this inhibition, prolyl and lysyl hydroxylase activities in the mast cells were less than 0.08% and the two hydroxylysyl glycosyltransferase activities less than 1% of those in the tendon cells. The intracellular enzyme pattern of collagen biosynthesis in the mast cells is thus completely or virtually completely repressed.     

Phospholipid metabolism of monkey smooth muscle cells grown in hyperlipemic serum.

The phospholipid content and synthesis of monkey smooth muscle cells grown in tissue culture with normal or hyperlipemic monkey serum were examined. The pattern of incorporation of radioactively labeled inorganic phosphate into the phospholipids of these cells was measured using a 4 h pulse of 32P. Phosphatidylcholine and phosphatidylinositol were the predominant phospholipids labeled. Although phosphatidylcholine constituted 45% of the cellular phospholipid, phosphatidylinositol had the highest specific activity. Exposure of the smooth muscle cells to hyperlipemic monkey serum did not alter the phospholipid content, composition or synthesis of these cells. The total phospholipid content of the smooth muscle cells was independent of the concentration of lipid in the media. The distribution of 32P into the phospholipids of monkey alveolar macrophages, L-cell mouse fibroblasts, and segments of the intima-media from monkey aortas is reported.     

The differentiation behaviour of MDCK cells grown on matrix components and in collagen gels

MDCK cells are grown on various substrates (Thermanox pure, extracellular matrix (ECM), dried or wet collagen type I or type III), on floating collagen and enclosed in collagen gels, and their differentiation behaviour is investigated electron microscopically. The cells grown on ECM or dried collagen (type I and type III) do not show any changes as compared with the controls (Thermanox). Differentiation processes can only be observed when the cells are grown on wet collagen (type I and type III), especially on floating collagen and enclosed in collagen gels. These differentiation processes comprise changes in the cell shape, an increase in the number of microvilli, an increase in the length of the lateral contact zone with the formation of gap junctions and desmosomes, and an increase in the number and size of the cell organelles. A basement membrane only develops in the form of short segments. Moreover, on floating collagen and in collagen gels three-dimensional, organoid structures develop: cell aggregates with central lumina and tubuli. They are formed by cuboid cells that also exhibit indications of differentiation. Basement membrane fragments occur more often and are longer. It can be concluded from these findings that the chemical structure of the substrate does not play the primary role in the described process. It is rather the physical properties, probably the plasticity, that are of significance. Due to this property the cells change their shape and the contact areas increase in size. The establishment of contacts might be the triggering factor for differentiation. Organoid structures with lumina develop when the apical surface comes into contact with other cells or collagen gels. The pronounced tendency towards polarization necessitates a re-arrangement of three-dimensionally growing cells to structures with lumina. The formation of the basement membrane is the result and not the cause of differentiation.