Chicken FK506-binding protein, FKBP65, a member of the FKBP family of peptidylprolyl cis-trans isomerases, is only partially inhibited by FK506.

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.

Calcium determines the shape of fibrillin.

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.

The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cell-specific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia.

Triple helix formation of procollagen type I can occur at the rough endoplasmic reticulum membrane.

One key problem in understanding the biosynthesis of collagens remains the assembly of the three alpha-chains. How and when are the different gene products selected, aligned, and folded into a triple helix? As the spatial arrangement during biosynthesis might be important, we concentrated on whether the rough endoplasmic reticular membrane is involved in this process. Microsomes were prepared from biosynthetically labeled chick tendon fibroblasts. Vesicles were spread as a monomolecular film which was then transferred over several compartments of a filmbalance containing fresh subphase. Fluorograms of the surface film showed that the monolayer contains procollagen chains. When the monolayer was transferred onto a chymotrypsin/trypsin-containing subphase, the gel bands of the proalpha-chains were shifted into the position of mature alpha-chains, indicating that only the propeptides were digested and the collagenous regions were protected due to triple helix formation. Our results suggest that newly synthesized proalpha-chains can associate as trimers and fold into a triple helical conformation while they are still associated with the membranes of the rough endoplasmic reticulum. These processes also occur when interchain disulfide linkage is inhibited, indicating that chain selection and registration is not dependent on formation of covalent bonds among the carboxyl propeptides.

Comparison of egg excretion and serum pepsinogen levels as measures of nematode worm burdens in calves with limited pasture exposure.

Four- to 6-mo-old calves raised on clean pastures were allowed to graze for about 30 days on pastures naturally contaminated with Ostertagia ostertagi, Cooperia spp., Haemonchus placei, and Nematodirus helvetianus. The calves were then housed on concrete for 3 wk before slaughter. At necropsy the eggs per gram of feces (EPG) and the total worm burdens from the abomasum and small intestine were determined. Blood samples were also obtained for serum pepsinogen (PEP) assays. A total of 44 calves was sampled over 27 mo. Adult worm totals were distributed more normally after logarithmic transformation (LOGTOTAL), and EPG and PEP were distributed more normally after square root transformation (SQRT--EPG; SQRT--PEP). Correlation between LOGTOTAL and either SQRT--EPG or SQRT--PEP was about 0.7. These correlations were higher than those obtained with nontransformed data, suggesting that either EPG or PEP are easily measured variables that explain a significant amount of the variation observed in total worm burdens. Polynomial regression models of a cubic order using the SQRT--EPG accounted for 78% of the variation observed in the LOGTOTAL, whereas SQRT--PEP accounted for 58% of the variance. Within the range of worm burdens observed, these results indicate that EPG has value in estimating total worm burdens of naturally infected calves, and that pepsinogen levels are of less value for analysis of total worm burden in calves with a limited previous exposure to trichostrongyle nematodes.

Thermal stability and folding of type IV procollagen and effect of peptidyl-prolyl cis-trans-isomerase on the folding of the triple helix.

Intact, monomeric type IV procollagen was isolated from the medium of PF-HR9 cells. Its stability was measured by optical rotatory dispersion, differential scanning calorimetry, and trypsin susceptibility of the partially unfolded molecules. At neutral pH, a complex transition between 35 and 42 degrees C and a smaller transition at 48 degrees C are observed by optical rotatory dispersion, using a heating rate of 10 degrees C/h. Reduction of the heating rate to 1.6 degrees C/h resulted in a 1 degree C lowering of the apparent melting temperatures. A similar curve is observed in 10 mM acetic acid, with transitions about 2 degrees C lower. Differential scanning calorimetry revealed transitions at 36.0, 42.1, and 48.0 degrees C at neutral pH, with a total transition enthalpy of 17.1 kJ/mol tripeptide units. In 10 mM acetic acid, transitions at 35.6, 38.9, 41.7, and 50.0 degrees C are observed. The transition enthalpy is 16.4 kJ/mol tripeptide units. The transition enthalpy is similar to values found for interstitial collagens. Results from trypsin digestion experiments are consistent with the stability found by optical methods and calorimetry. The rate and completeness of refolding after melting were measured. In neutral buffer, the initial rate was found to be 0.041 min-1, faster than the refolding rates observed with types pN III and III collagen. Peptidyl prolyl cis-trans-isomerase increased the refolding rate to 0.083 min-1, indicating that cis-trans-isomerization is the rate-limiting step, despite the interruptions in the triple helix. Trypsin digestion experiments indicated that the refolding mechanism is similar in the presence and absence of the enzyme. Refolding was nearly complete in neutral buffer. In 10 mM acetic acid, folding was considerably slower and went to about 74% completion. In both solvents, the refolded material was only slightly less stable than the native material. Electron microscopy of partially refolded samples showed that most refolding started at the COOH terminus, but some was initiated at other sites.

Role of a membrane glycoprotein in Friend virus-induced erythroleukemia: studies of mutant and revertant viruses.

We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. Natl. Acad. Sci. USA 80, 4704-4708). In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein structure and localization which are essential for its leukemogenic activity.

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity