Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding

Abstract

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

Keywords: Collagen; Connective Tissue; Ehlers-Danlos Syndrome; Glycine Mutations; Mutant; Prolyl Isomerase; Protein Folding; Type III Collagen.

FIGURE 1.

The sequence of the designed human type III collagen C-terminal quarter fragment with (GPP)₇ and the type XIX collagen NC2 domain. 252 (775–1026) residues of the human type III collagen C-terminal end were used for the experiments schematically shown in A. The Pro-1026 is the last tripeptide unit before the cystine knot sequence -Gly-Pro-Cys-Cys-Gly-Gly- in the natural sequence. In the designed fragment the cystine knot is replaced by the (Gly-Pro-Pro)₇-NC2 domain of type XIX collagen. (GlyProPro)₇ was attached to align the three staggered chains properly. The positions of the mutated residue Gly-910 and Gly-1018 are indicated in bold. The complete sequence of the expressed quarter fragment is shown in B.

FIGURE 2.

Rotary shadowing images of the QF. The purified QF protein (0.1 mg/ml) was dialyzed against 100 mm ammonium bicarbonate at 4 °C, sprayed onto mica, and shadowed with platinum (34). The images were viewed by transmission electron microscopy. A representative field is shown in A. Only molecules with defined, extended tails were measured. The observed average length of the strings is 73.8 nm (S.D. 6.9 nm). Statistical analysis was performed, and a S.D. value was calculated. The length distribution is shown in B.

FIGURE 3.

Thermal transition curves of the QFs. The samples were run in 20 mm Tris/HCl buffer, pH 7.0, containing 200 mm NaCl and 0.05% Tween 20. Samples were equilibrated at 4 °C for more than 2 days and heated with a rate of 10 °C/h. The CD signal at 223 nm was monitored. At 35 °C the heating was stopped for 10 min to achieve complete unfolding of the QF. The temperature was then lowered at a rate of 10 °C/h. The lines are colored as follows: WT (black), G910A (red), G910V (blue) shown in A and WT (black), G1018A (green), and G1018V (violet) shown in B. The arrows indicate the direction of the temperature change.

FIGURE 4.

Trypsin digestion of the QFs. A, the QF sample solutions were incubated at 15, 20, or 33 °C without trypsin (0) or for 5 or 10 min (5, 10) with trypsin (8 μg/ml at final concentration). After the 12% SDS-PAGE, the proteins were blotted onto a membrane and subjected to Edman degradation. Blots stained with GelCode Blue are shown. B, trypsin-resistant peptides identified by N-terminal peptide sequencing are indicated by arrows with the residue number. Lys and Arg sites are indicated as vertical lines above and below the horizontal line, respectively. The potential cleavage sites for trypsin are as follows: Lys-810, -815, -864, -893, -927, -939, and -983, and Arg-789, -801, -825, -845, -857, -867, -897, -915, -924, -933, -942, -972, -999, -1002, and -1005. Mutation sites are marked with big arrows.

FIGURE 5.

Refolding kinetics of the QF by CD. The QF proteins were unfolded at 33 °C for 10 min. Refolding at 4 °C was monitored at 223 nm by CD. The traces are for WT (black), G910A (red), G910V (blue), G1018A (green), and G1018V (violet). Shown are the long term experiments (A) and the first 1000-s periods (B) with the linear fits used to calculate the initial first order rates in Table 1.

FIGURE 6.

Refolding kinetics of the QF analyzed by trypsin resistance of folded fragments. QFs of WT, G910A, G910V, G1018A, and G1018V proteins were unfolded at 33 °C for 10 min. The refolding of the triple helix was monitored by trypsin cleavage resistance of the folded helical portions. The unfolded collagen is readily cleaved by trypsin, whereas the refolded triple helical domain is resistant. The time points indicated are when the trypsin solution was added to the sample. After the 2-min incubation on ice, the enzyme was inactivated. A, the proteins were analyzed by 12% SDS-PAGE followed by GelCode Blue staining. The time point is indicated in minutes unless specified as hours. St, starting material; E, trypsin; +E, trypsin-treated sample without heat denaturation; M, protein marker. Trypsin shows two bands indicated as arrows in the WT panel. The molecular weight of the globular protein markers is indicated in kDa. B, the gel of WT was scanned, and the relative abundance of several bands was plotted. The bands are as indicated on the first gel (WT). The relative abundance of the e band (fully folded chains) of all peptides was quantitated and is shown in C.

FIGURE 7.

Simple model to fit the refolding curves. A, the C-terminal QF of type III collagen is trimerized by (GPP)₇-type XIX collagen NC2 domain (XIX NC2). After heat treatment at 33 °C, the type III collagen part is unfolded, but (GPP)₇ keeps three chains at the correct stagger. Refolding starts at the C terminus of QF. For the peptides with Gly-910 mutation the refolding trace is about the same as for WT until the residue Gly-910 is encountered. At the mutated site, the renucleation of the triple helix is necessary to restart the triple helix formation. The mutated peptides with the Val mutations take longer time to renucleate than those with the Ala mutations. For the Gly-1018 peptides the initial refolding is affected from the very beginning. Once the triple helix is renucleated after the Gly-1018 site, the zipper-like folding proceeds with the same rate. B, the model used for the fitting is illustrated. Details are described in the text. All the reaction steps are assumed as the first order. The folding rate for the helix fragment without a mutation is defined as k_n. The renucleation rate (without propagation of helices) at the site of a mutation is defined as k_m. The vertical line in the middle of QF indicates the point of a half-conversion. C, shown are experimental and fitted curves. The experimental curves are the same as in Fig. 5A. A single parameter k_m,A was used for the Ala mutants or k_m,V for the Val mutants. The experimental data are plotted as circles, WT (black), G910A (red), G910V (blue), G1018A (green), and G1018V (violet). The fitted curves are indicated as lines with the corresponding color. D, all k_m parameters were fitted individually for each mutant. The same colors are used as in C.