Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.     

Expression and localization of collagen-binding stress protein Hsp47 in mouse embryo development: comparison with types I and II collagen

Heat shock protein (Hsp)47 is a collagen-binding stress protein localized in the endoplasmic reticulum and is thought to have chaperone-like functions that are specific to procollagen biosynthesis. In previous papers, we reported that the expression of Hsp47 is closely correlated with that of various types of collagen in various cell lines and also in the progression of experimental liver fibrosis. In the present study, the expression of Hsp47 was examined during the development of mouse embryos by immunostaining with an anti-Hsp47 antiserum. The spatio-temporal correlation of the expression of Hsp47 with those of types I and II collagen was also examined using specific antisera. Hsp47 expression during embryogenesis was observed mainly in mesoderm and in tissues that are derived from mesoderm, such as connective tissue, cartilage, bone, notochord and somites. Hsp47 was also detected in tissues derived from the neural crest mesenchyme. In the central nervous system, Hsp47 was detected in some restricted regions where cells proliferate, such as the ventral area of the neural tube and choroid plexus. Immunostaining for types I and II collagen revealed the spatial and temporal correlations of the expression of these proteins with that of Hsp47. These results suggest the biological importance of Hsp47 as a collagen-specific molecular chaperone in the mouse developmental program.     

Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides

Peptides prepared from the amino termini of pro alpha 1(I) and pro alpha 1(III) collagen chains inhibit the production of pro alpha 1(I) and pro alpha 2 by rat calvaria rna in a reticulocyte cell-free system. The synthesis of other proteins was not altered, suggesting a specific effect on collagen production. Various peptides from the helical region of the alpha 1(I) chain did not alter translation. These studies, taken together with earlier studies showing inhibition of collagen synthesis by cells in culture receiving the amino-terminal peptides, are consistent with a regulatory function in collagen synthesis for the amino-terminal peptides from procollagen.     

Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.     

Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen

Hsp47 is a heat-shock protein that interacts transiently with procollagen during its folding, assembly and transport from the endoplasmic reticulum (ER) of mammalian cells. It has been suggested to carry out a diverse range of functions, such as acting as a molecular chaperone facilitating the folding and assembly of procollagen molecules, retaining unfolded molecules within the ER, and assisting the transport of correctly folded molecules from the ER to the Golgi apparatus. Here we define the substrate recognition of Hsp47, demonstrating that it interacts preferentially with triple-helical procollagen molecules. The association of Hsp47 with procollagen coincides with the formation of a collagen triple helix. This demonstrates that Hsp47's role in procollagen folding and assembly is distinct from that of prolyl 4-hydroxylase. These results indicate that Hsp47 acts as a novel molecular chaperone, potentially stabilizing the correctly folded collagen helix from heat denaturation before its transport from the ER.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Direct in vitro and in vivo evidence for interaction between Hsp47 protein and collagen triple helix

Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis

Heat-shock protein of 47 kDa (Hsp47) is a molecular chaperone that recognizes collagen triple helices in the endoplasmic reticulum (ER). Hsp47-knockout mouse embryos are deficient in the maturation of collagen types I and IV, and collagen triple helices formed in the absence of Hsp47 show increased susceptibility to protease digestion. We show here that the fibrils of type I collagen produced by Hsp47-/- cells are abnormally thin and frequently branched. Type I collagen was highly accumulated in the ER of Hsp47-/- cells, and its secretion rate was much slower than that of Hsp47+/+ cells, leading to accumulation of the insoluble aggregate of type I collagen within the cells. Transient expression of Hsp47 in the Hsp47-/- cells restored normal extracellular fibril formation and intracellular localization of type I collagen. Intriguingly, type I collagen with unprocessed N-terminal propeptide (N-propeptide) was secreted from Hsp47-/- cells and accumulated in the extracellular matrix. These results indicate that Hsp47 is required for correct folding and prevention of aggregation of type I collagen in the ER and that this function is indispensable for efficient secretion, processing, and fibril formation of collagen.     

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.     

Endoplasmic reticulum protein HSP47 binds specifically to the N-Terminal globular domain of the amino-propeptide of the procollagen I α1(I)-chain

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with³⁵[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)-N-propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro α1 (l)-N-propeptide domain were confirmed by examination of polysome-associated pro α chains. The nascent pro α1(l)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro α1 (l)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)-N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro α1(l)- and pro α2(l)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)-chains and hsp47, whereas there was strong interaction between pro α1 (l)-chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino-propeptide region in particular. © 1995 Wiley-Liss, Inc.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

针对I型及Ⅲ型前胶原基因的二联核酶的构建及体外活性研究

增殖性瘢痕组织中胶原蛋白的合成显著增加从而导致胶原的过度沉积。利用核酶特异地抑制前胶原基因的表达可减少胶原蛋白的合成,为瘢痕的研究和防治提供了新的思路。为研究用核酶抑制前胶原基因表达的可能及效果,设计并构建了针对α1（I）型及α1（Ⅲ）型前胶原基因的二个单价核酶串联的二联核酶基因真核表达载体,并对其体外切割活性 进行研究。结果表明该二联核酶的切割效果明显,均能有效地切割底物,为进一步研究核酶的前胶原基因表达的抑制作用以及利用核酶防治瘢痕产生打下基础。     

Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III)

Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis. Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison. Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents. This decrease was specific for collagenous proteins. From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro alpha 1(I) molecules but that their pro alpha 1(I) migrated slightly faster than the parental and control counterparts. Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage. Upon pepsin treatment, extracellular alpha 1(I) and alpha 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro alpha 1(I) chains was related to COOH or NH2 terminal propeptides. In agreement with the reduced type I collagen production, an increase in the alpha 1(III)/alpha 1(I) ratio was also detected. Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro alpha 2(I) and alpha 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro alpha 1(I)/pro alpha 2(I) that was measured after dithiothreitol reduction. Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro alpha 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients. Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts. Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta. Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation. Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested.     

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture.

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.