Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are idubited by cyclosporin A with a Ki of 25 -50 pM, a concentration 1000-fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Aka-Pro-Phe-methyl-coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type 111 collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% a-helix, 34% B-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.

The chicken FK506-binding protein FKBP65, a peptidylprolyl cis-trans isomerase, is a rough endoplasmic reticulum protein that contains four domains homologous to FKBP13, another rough endoplasmic reticulum PPIase. Analytical ultracentrifugation suggests that in FKBP65 these four domains are arranged in a linear extended structure with a length of about 26 nm and a diameter of about 3 nm. All four domains are therefore expected to be accessible to substrates. The specificity of FKBP65 towards a number of peptide substrates was determined. The specific activity of FKBP65 is generally lower than that of FKBP12 when expressed as a per domain activity. The substrate specificity of FKBP65 also differs from that of FKBP12. Inhibition studies show that only one of the four domains can be inhibited by FK506, a powerful inhibitor of all other known FKBPs. Furthermore, the same domain seems to be susceptible to inhibition by cyclosporin A. No other FKBPs were shown to be inhibited by cyclosporin A. It is also shown that FKBP65 can catalyse the re-folding of type III collagen in vitro with a kcat/Km = 4.3 x 10(3) M-1.s-1.

HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens. Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K half ) of 1.4 ؋ 10 ؊7 M, and a Hill coefficient of 4.3 was observed. Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli. Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker. Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed. Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix. Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.HSP47 (47-kDa heat shock protein) is a rough endoplasmic reticulum protein believed to function as a collagen-specific molecular chaperone (1-3). It was shown that HSP47 (also known as colligin, J6, gp46, and CB48) is co-expressed with collagens in cells such as fibroblasts and chondrocytes (4,5). In mouse embryo development, HSP47 is expressed mainly in mesoderm and mesoderm-derived tissues, and the expression correlates both temporally and spatially with that of type I and II collagen (6). HSP47 knockout mice show an embryonic lethal phenotype (7). HSP47 is isolated easily by affinity chromatography on gelatin-Sepharose (8, 9) and was later shown to interact also with triple helical collagen types I-V (10). It was found that all these collagens have a similar affinity for HSP47 with dissociation constants of about 10 Ϫ7 M (10). The interaction of HSP47 with both unfolded and folded procollagens is unusual and distinguishes HSP47 from other molecular chaperones.A number of potential functions for HSP47 during procollagen biosynthesis have been described. HSP47 binds to nascent procollagen chains and therefore may help prevent the premature association of procollagen chains and/or assist with the translocation of procollagen chains into the rough endoplasmic reticulum (11). HSP47 also interacts with procollagen chains in the rough endoplasmic reticulum when triple helix formation is inhibited by ␣,␣Ј-dipyridyl, an inhibitor of prolyl-4-hydroxylase (9). A potential role in vesicular trafficking was explored with Brefeldin A and monensin (9,12,13). In Brefeldin A-treated cells HSP47 remained associated with procollagen, whereas in cells treated with monensin no HSP47 binding was detected.Together this shows that HSP47 seems to release procollagen on entry to the Golgi. Procollagens are not secreted as individual molecules, but ...

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