Ziploc-ing the structure 2.0: Endoplasmic reticulum-resident peptidyl prolyl isomerases show different activities toward hydroxyproline

Abstract

Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER.

Keywords: biosynthesis; collagen; endoplasmic reticulum (ER); molecular chaperone; post-translational modification (PM); prolyl isomerase.

Figure 1.

SDS-PAGE gel of purified rER-resident PPIases. The purified rER-resident PPIases were run on NuPAGE Novex BisTris 4–12% gel (Thermo Fisher Scientific) and stained with GelCode Blue Stain Reagent. Enzyme names shown are: CypB, recombinant chicken CypB; FKBP13, recombinant human FKBP13; FKBP19, the FKBP domain of recombinant human FKBP19; FKBP22, recombinant human FKBP22; FKBP23, recombinant human FKBP23; FKBP65, endogenous chicken FKBP65.

Figure 2.

Circular dichroism measurements of FKBPs. The wavelength scan and thermal transition were measured using circular dichroism. A, the wavelength spectra of recombinant human FKBP13 (filled circle) and FKBP domain of recombinant human FKBP19 (open circle). B, the wavelength spectra of recombinant human FKBP22 (filled circle) and recombinant human FKBP23 (open circle). C, the wavelength spectra of recombinant human FKBP23 in presence (filled circle) or absence (open circle) of calcium. D, the thermal transition curves of recombinant human FKBP13 (filled circle) and FKBP domain of recombinant human FKBP19 (open circle). E, the wavelength spectra of recombinant human FKBP22 (filled circle) and recombinant human FKBP23 (open circle). The FKBP domain of FKBP19 is indicated as FKBP19 in the figures.

Figure 3.

Molecular chaperone function of FKBPs. A and B show the classical chaperone activity assay using the thermal aggregation of citrate synthase. The assay was monitored at 500 nm, and 30 μm citrate synthase solution was diluted 200-fold into prewarmed 40 mm HEPES buffer, pH 7.4, containing 1 mm CaCl₂ at 43 °C. A, in the absence (black) and presence (red) of recombinant human FKBP13, recombinant human FKBP domain of FKBP19 (blue), and recombinant human FKBP23 (green). The protein concentration was 1.0 μm for these FKBPs. 0.1 μm PDI was used as a positive control (magenta). All curves are averaged by a minimum of three measurements. B, results are shown as the percentage of inhibitions of thermal aggregation of citrate synthase with 0.5, 1.0, and 3.0 μm concentrations of the FKBP domain of FKBP19. The amount of aggregates generated without the FKBP domain of FKBP19 was set as 100%, and 0.1 μm PDI is shown as a comparison. Each data point was calculated by a minimum of three measurements using three independent experiments. C and D show the fibril formation of type I collagen. A stock solution of type I collagen in 50 mm acetic acid was diluted to a final concentration of 0.1 μm. C, the measurements were performed in 0.1 m sodium bicarbonate buffer, pH 7.8, containing 0.15 m NaCl and 1 mm CaCl₂ at 34 °C. Shown in the absence (black) and presence (red) of recombinant human FKBP13, recombinant human FKBP domain of FKBP19 (blue), and recombinant human FKBP23 (green). The protein concentration was 0.5 μm for these FKBPs. 0.1 μm decorin was used as a positive control (magenta). All curves are averaged by a minimum of three measurements. D, the measurements were performed in 0.15 m sodium phosphate buffer, pH 7.8, containing 0.15 m NaCl at 34 °C. Shown is the absence (black) and presence (red) of 0.5 μm, 1.0 μm (blue), and 3.0 μm (green) recombinant human FKBP domain of FKBP19. 0.1 μm decorin was used as a positive control (magenta). All curves are averaged by a minimum of three measurements. The FKBP domain of FKBP19 is indicated as FKBP19 in the figures.

Figure 4.

Refolding of the carboxyl-terminal quarter fragments of type III collagen without prolyl 4-hydroxylation and full-length type III collagen. Kinetics of the refolding of the carboxyl-terminal quarter fragment of type III collagen without prolyl 4-hydroxylation (final concentration 2.0 μm) and full-length type III collagen (final concentration 0.2 μm) in the presence of the rER-resident PPIases monitored by CD at 220 nm is shown. The protein concentrations used were 3.0 μm and 2.0 μm for FKBP13, 6.0 μm and 2.0 μm for the FKBP domain of FKBP19, FKBP22, and FKBP23, 3.0 μm and 1.0 μm for FKBP65, and 0.5 μm and 1.0 μm for CypB for the carboxyl-terminal quarter fragment of type III collagen without prolyl 4-hydroxylation and full-length type III collagen, respectively. All curves are averaged by a minimum of three measurements. A–B and C–D indicate the refolding of the carboxyl-terminal quarter fragments of type III collagen without prolyl 4-hydroxylation and the refolding of full-length type III collagen, respectively. The absence of PPIases is shown as black in A–D. The presence of PPIases is annotated in individual figures using red, blue, and green. The FKBP domain of FKBP19 is indicated as FKBP19 in the figures.

Figure 5.

Catalytic efficiency for the prolyl isomerization using pNa peptide substrates. The catalytic efficiency (k_cat/K_m) for the isomerization reaction was determined by kinetic measurements using the Suc-Ala-XY-Phe-pNa peptide substrates, where XY indicates the combination of two amino acids labeled in graphs. The enzymatic activity of prolyl isomerization of seven peptide substrates for six PPIases, including the mean ± S.D. as a magenta line derived from Table 3, is indicated. The p value is annotated as follows (*, p < 0.05; **, p < 0.005; ***, p < 0.0005. NS, not significant). The FKBP domain of FKBP19 is indicated as FKBP19.