Supramolecular Organization of the α121-α565 Collagen IV Network^*

Isolation of α1256 NC1 Hexamer from Aorta

Aorta NC1 hexamers were prepared as described previously by Kahsai et al. (20). Briefly, NC1 hexamers were solubilized from bovine aorta tissue by the collagenase digestion method and purified on DE-52 cellulose and Sephacryl S-300 columns.

Separation of the aorta NC1 hexamers of different α-chain composition was performed in an ÄKTA purifier HPLC (GE Healthcare) with a Mono S cation exchange column (GE Healthcare). Aorta NC1 hexamers were loaded into the Mono S column and eluted with a linear gradient of increasing concentrations of NaCl; absorbance was monitored at 280 nm. Each chromatographic peak was analyzed by Western blotting with monoclonal antibodies H11 (anti-α1 NC1 domain), H22 (anti-α2 NC1 domain), H31 (anti-α3 NC1 domain), H44 (anti-α4 NC1 domain), H52 (anti-α5 NC1 domain), and B66 (anti-α6 NC1 domain) (21). Fractions staining for α1, α2, α5, and α6 NC1 domains were pooled and stored at −20 °C.

Mass Spectrometry Analysis of NC1 Dimer Peptides Derived from α1256 NC1 Hexamer

The fraction containing the α1256 hexamer (Mono S peak 2) was denatured and reduced by mixing equal volumes of protein solution with a 0.4 m Tris-HCl, pH 7.5, buffer containing 8 m guanidine-HCl and 50 mm dithiothreitol. After heating samples in a boiling water bath for 10 min, the proteins were alkylated with 50 mm iodoacetamide in the dark for 30 min at room temperature. After ethanol precipitation at −20 °C for 2 h, NC1 subunits were resuspended in 0.1 m ammonium bicarbonate, pH 7.5, and digested with trypsin overnight. Subsequently, tryptic peptides were fractionated on a Superdex Peptide column (GE Healthcare), and polypeptides from 3000 to 6500 Da were collected, lyophilized, and stored at −20 °C before MS analysis.

For mass spectrometric analysis of sulfilimine-cross-linked peptides, dry samples were reconstituted in 0.1% formic acid and loaded onto a capillary reverse phase analytical column (360 μm outer diameter × 100 μm inner diameter) using an Eksigent NanoLC Ultra HPLC and autosampler. The analytical column was packed with 20 cm of C18 reverse phase material (Jupiter, 3 μm beads, 300 Å, Phenomenex) equipped with a laser-pulled emitter tip. Peptides were gradient-eluted at a flow rate of 500 nanoliters/min, and the mobile phase solvents consisted of 0.1% formic acid, 99.9% water (solvent A) and 0.1% formic acid, 99.9% acetonitrile (solvent B). A 90-min gradient was performed and consisted of the following: 0–10 min, 2% B; 10–50 min, 2–35% B; 50–60 min, 35–90% B; 60–65 min, 90% B; 65–70 min, 90–2% B; 70–90 min, 2% B. Upon gradient-elution, peptides were mass analyzed on a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA), equipped with a nanoelectrospray ionization source. The instrument was operated using a data-dependent method where full scan (m/z 400–2000) spectra were acquired with the Orbitrap as the mass analyzer (resolution 60,000), and the three most abundant ions in each MS scan were selected for collision-induced dissociation (CID) fragmentation in the LTQ Velos ion trap, which were followed by data-dependent MS³ analysis of the three most abundant ions in each MS² scan. An isolation width of 2 m/z, activation time of 30 ms, and 35% normalized collision energy were used to generate MS² and MS³ spectra. For MS² scans, a minimum threshold of 500 was used to trigger data-dependent spectra, whereas a threshold of 500 or 100 was used to trigger MS³ spectra. Dynamic exclusion was enabled, with an exclusion duration time of 20 ms.

Sulfilimine Cross-linking Analysis of NC1 Peptide Mass Spectra Data

Mass spectra analyses were performed both manually with Xcalibur 2.1 and computationally with the proteomic analysis software SEQUEST (22, 23) and/or MyriMatch (24). For manual searches, theoretical peptide masses for NC1 peptides containing either Met⁹³ or Lys²¹¹ were generated using GPMAW software (Lighthouse Data). Sulfilimine-cross-linked peptide masses were calculated by subtracting 2 hydrogen atoms from the sum of the masses of Met⁹³- and Lys²¹¹-containing peptides. Although we hypothesized that the α1256 hexamer results from the interaction of a α121 trimer with a α565 trimer, forming α1-α5 and α2-α6 NC1 dimers, we did not limit our analyses to these combinations and also considered other possible combinatorial arrangements. Sulfilimine-cross-linked peptide complexes were manually searched for using the QualBrowser of Xcalibur 2.1 software.

For sulfilimine-cross-linked peptides not identified manually, we took advantage of the characteristic CID fragmentation of sulfilimine-cross-linked peptides (9), which generates modified Met and Lys residues, to design an alternative unbiased bioinformatics approach to analyze mass spectra. Proteomic analysis software Scaffold 3 (Proteome Software, Portland, OR) was used to compile SEQUEST and/or MyriMatch searches of the bovine subset of the Uniref100 database for sulfilimine cross-link-derived peptides. The MyriMatch algorithm (24) was run on a two six-core Intel Xeon processor HP Proliant DL160 G6 server running Windows server 2008 R2 enterprise using the Bumbershoot suite (25). The search was adapted for identifying peptides with the following amino acid modifications: variable, −48.0034 on Met, +45.9877 or +61.9826 on Lys, and oxidation of methionine (+15.9949) as well as fixed carbamidomethylation of cysteine (+57.021). The proteomic analysis software ScanRanker was used to assess the MS³ spectral quality of peptide sequences containing Met⁹³ or Lys²¹¹ (26). Spectra were annotated and validated with the utility program IonMatcher (24).

Homology Modeling of the α121-α565 Hexamer

A homology model of the α121-α565 NC1 hexamer was constructed with SWISS-MODEL server and visualized with UCSF Chimera (27, 28). The crystal structure of bovine α121 hexamer (Protein Data Bank accession code 1T61) was used as the starting template (17). The sequences of bovine α5, α5, and α6 NC1 domains were aligned with bovine α1, α1, and α2 NC1 sequences, respectively, and then threaded onto the α121 trimer template (28). In cases where there were deletions in the primary sequence, the sequence variations were reiteratively modeled and stereochemically refined. The model with the lowest root mean square deviation was retained. The α565 trimer model was superimposed “head-to-head” with the bovine α121 trimer crystal structure to form the α1256 hexamer model. Molecular graphics images were produced using UCSF Chimera 1.6.1.rc and ray-traced with POV-Ray (Version 3.6) (27).

Isolation and Characterization of α1256 Hexamer from Bovine Aorta

The α1256 hexamer was isolated from bovine aorta tissue to provide NC1 peptides amenable to analysis by mass spectrometry. Digestion of the aorta tissue with bacterial collagenase routinely yielded about 1.5 mg of NC1 hexamers per 50 g of wet tissue (Fig. 1). In accord with previous results (16), immunoblot analysis using chain-specific monoclonal antibodies confirmed the presence of the α1, α2, α5, and α6 NC1 domains in the purified aorta NC1 hexamer (Fig. 1B). Notably, α3 and α4 NC1 domains were not detected. Monoclonal antibodies detected NC1 dimeric subunits, suggesting the presence of covalent cross-linking between any combination of α1, α2, α5, and α6 NC1 monomeric subunits.

In smooth muscle tissues two distinct subpopulations of NC1 hexamers of different α-chain compositions were observed: the α121 and α1256 hexamers (16). To isolate the α1256 hexamer for MS analysis, the preparation of aorta NC1 hexamers was fractionated on a cation-exchange Mono S chromatography column (Fig. 2A). Elution of bound proteins with increasing concentrations of sodium chloride yielded three distinct chromatographic peaks. Immunoblot analysis of each peak using chain-specific monoclonal antibodies showed the α5 and α6 NC1 subunits eluting most abundantly in peaks 1 and 2, with the α1 and α2 NC1 subunits also found in these peaks (Fig. 2B). Peak 3 is predominantly composed of α1 and α2 NC1 subunits, whereas the staining for α5 and α6 NC1 domains was minimal. These data indicate that the α121 hexamer elutes in peak 3, whereas the α1256 hexamer elutes in peaks 1 and 2. Peak 1 and 2 were considered indistinguishable by immunoblot analyses, but because of its greater abundance, peak 2 was used for MS analyses.

Mass Spectrometry Analysis of Sulfilimine-Cross-linked NC1 Dimer Peptides

NC1 hexamers are formed by the interaction of the C-terminal NC1 domain trimers of two protomers (17). The large, planar interface formed by the interacting trimers is stabilized by sulfilimine bonds (SN) in which the sulfur atom of Met⁹³ from an NC1 monomer forms a covalent bond with the ϵ-nitrogen from Lys²¹¹ (or hydroxylysine) of an adjacent NC1 monomer (9, 29). Because of the spatial alignment of NC1 domains across the trimer-trimer interface, the occurrence of sulfilimine bonds results in the formation of three covalently linked NC1 dimers (Fig. 3). Therefore, the composition of such sulfilimine-cross-linked NC1 dimers reflects the way in which triple-helical protomers interact to form collagen IV networks.

In this study we used a MS-based strategy to determine the occurrence of sulfilimine bonds by identifying cross-linked peptides derived from NC1 dimeric subunits. Thus, as previously suggested (16), we hypothesized that this hexamer is formed from a α121 trimer and a α565 trimer. This arrangement may generate up to four distinct sulfilimine cross-linking sites: two sites between α1 and α5 NC1 domains and two sites between α2 and α6 NC1 domains (Fig. 3A). Theoretical masses for α1-α5 and α2-α6 sulfilimine-cross-linked NC1 peptides as well as other possible combinations were calculated as indicated under “Experimental Procedures.” To identify these sulfilimine-cross-linked peptides, Mono S peak 2, containing the α1256 NC1 hexamer (Fig. 2), was digested with trypsin, and the resulting peptides were fractionated by gel filtration (Fig. 3B). Peptides ranging from 3000 to 6500 Da in size were collected and analyzed in a high resolution LTQ Orbitrap Velos in a data-dependent manner as previously described (9, 18). For convenience, sulfilimine-cross-linked peptides identified by manual and bioinformatics analyses of MS data described under “Experimental Procedures” are summarized and presented by the type of NC1 dimer from which they originate.

Mass Spectrometry Analyses of α1-α5 NC1 Sulfilimine-Cross-linked Peptides

The α1-Hyl²¹¹−α5-Met⁹³ complex was found in the full scan mass spectrum containing the multiply protonated ions (+4, +5, +6) that represent the T-3958 complex (Fig. 4A). The difference between observed and calculated masses for this peptide complex reflects the loss of 2 hydrogen atoms upon sulfilimine bond formation (Table 1). To confirm the presence of a sulfilimine bond connecting the two peptides, the m/z 792.5850 (+5) ion was selected for MS² fragmentation by CID. As observed in the MS² spectrum (Fig. 4A), CID fragmentation yielded two predominant ions: m/z 834.3 (+3) and m/z 730.7 (+2), which represent the α5-Met⁹³- and α1-Hyl²¹¹-containing peptides but deviate from the expected theoretical peptide masses by −48 and +46 atomic mass units, respectively. These peptide modifications correspond to the olefin fragment derived from Met⁹³ and the methylsulfenamide fragment derived from Hyl²¹¹ that result from the characteristic gas-phase Cope elimination previously described for sulfilimine-cross-linked peptides (supplemental Figs. S1 and S2) (9). The T-3958 peptide complex demonstrates hydroxylation of α1-Lys²¹¹ and establishes the presence of the sulfilimine bond in one of the two cross-linking sites within the α1-α5 NC1 dimer.

Similarly for the α1-Met⁹³−α5-Hyl²¹¹ NC1 peptide complex, the full scan mass spectrum contained multiply protonated ions (+5 and +6) representing the T-4943 complex (Fig. 4B). The difference between the observed and calculated masses for this peptide complex reflects the loss of 2 hydrogen atoms upon sulfilimine bond formation (Table 1). To confirm the presence of a sulfilimine bond connecting the two peptides, the m/z 824.9004 (+6) ion was selected for MS² fragmentation by CID. As observed in the MS² spectrum (Fig. 4B), CID fragmentation yielded two predominant ions; m/z 689.1 (+2) and m/z 893.6 (+4) ions represent the α5-Hyl²¹¹- and α1-Met⁹³-containing peptides but deviate from the expected theoretical peptide masses by +46 and −48 atomic mass units and, respectively, which is consistent with a MS² fragmentation profile characteristic of peptides cross-linked by a sulfilimine bond (9). Furthermore, MS³ fragmentation confirmed the NC1 peptide sequences and localized the peptide modifications to Met⁹³ and Hyl²¹¹ (supplemental Figs. S3 and S4). Mass spectrometric analyses of the T-4943 peptide complex demonstrates hydroxylation of α5-Lys²¹¹ and establishes the presence of the sulfilimine bond in the second of the two α1-α5 NC1 dimer cross-linking sites.

Mass Spectrometry Analyses of α2-α6 NC1 Sulfilimine-Cross-linked Peptides

Although the hydroxylation of α6-Lys²¹¹ had never been reported, we included this modification in our mass spectra searches because this residue lies embedded within a conserved hydroxylation motif (30). The α2-Met⁹³−α6-Hyl²¹¹ NC1 peptide complex was found in the full scan mass spectrum containing the multiply protonated ions (+5, +6, +7) that represent the T-5307 complex (Fig. 5A). The difference between the observed and calculated masses reflects the loss of two hydrogen atoms upon sulfilimine bond formation (Table 1). As shown in Fig. 5A, CID fragmentation of m/z 885.4390 (+6) yielded three predominant ions in the MS² spectrum: m/z 773.4 (+3) and m/z 767.4 (+3) ions and an m/z 998.9 (+3) ion. These ions represent the α6-Hyl²¹¹- and α2-Met⁹³-containing peptides but deviate from the expected theoretical peptide masses by +62 and −48 atomic mass units, respectively, generating a MS² fragmentation profile characteristic of peptides cross-linked by a sulfilimine bond (9). MS³ fragmentation profiles of the m/z 767.4 (+3) and m/z 998.9 (+3) ions confirmed the NC1 peptide sequences and localized the peptide modifications to Met⁹³ (supplemental Figs. S5 and S6). Furthermore, MS³ validation with IonMatcher localizes the addition of 16 atomic mass units to Lys²¹¹ of the α6 NC1 domain (supplemental Fig. S5). These data demonstrate the hydroxylation of α6-Lys²¹¹ and establishes the occurrence of a sulfilimine bond in one of the two cross-linking sites within the α2-α6 NC1 dimer.

Similarly for the α6-Met⁹³−α2-Hyl²¹¹ NC1 peptide complex, the full scan MS1 mass spectrum contained multiply protonated ions (+3, +4, +5) representing the T-4393 complex (Fig. 5B). The difference between the observed and calculated mass for this peptide complex reflects the loss of 2 hydrogen atoms upon sulfilimine bond formation (Table 1). To confirm the presence of a sulfilimine cross-link, the m/z 1099.3181 (+4) ion was selected for MS² fragmentation by CID. As observed in the MS² spectrum (Fig. 5B), CID fragmentation yielded two predominant ions; m/z 731.7 (+2) and m/z 1468.4 (+2) ions represent the α2-Hyl²¹¹- and α6-Met⁹³-containing peptides but deviate from the expected theoretical peptide masses by +46 and −48 atomic mass units, respectively, which is consistent with a MS² fragmentation profile characteristic of peptides cross-linked by a sulfilimine bond (9). Furthermore, MS³ fragmentation confirmed the NC1 peptide sequences and localized the modifications to Met⁹³ and Hyl²¹¹ (supplemental Figs. S7 and S8). Collectively, these results demonstrate hydroxylation of α2-Lys²¹¹ and establish the presence of the sulfilimine bond in the second of two cross-linking sites in the α2-α6 NC1 dimer, which along with the four α1-α5 sulfilimine bonds establishes the presence of a total of six sulfilimine bonds per α121-α565 NC1 hexamer. Taken together, our MS studies of NC1 heterodimers support our hypothesis that the α1256 hexamer is formed by the covalent interaction of α121 trimer and the α565 trimer.

Homology Modeling of the α121-α565 Hexamer

Identification of sulfilimine-cross-linked NC1 dimers establishes the spatial arrangement and connectivity of the NC1 domains at the trimer-trimer interface and provides the framework to construct a three-dimensional homology model of the α121-α565 hexamer quaternary structure. The amino acid sequences of α1, α2, α5, and α6 NC1 domains were threaded onto the α121 NC1 hexamer crystal structure template, as described under “Experimental Procedures.” As shown in Fig. 6,⁴ the overall structure of the homology model for α1256 hexamer is very similar to the already established α121 and α345 hexamer. The model illustrates the interaction of a α121 trimer with a α565 trimer in which the orientation of one with respect to the other is determined by the alignment of α1-α5 and α2-α6 NC1 dimers revealed by the MS analysis of tryptic peptides identified in this study. Henceforth, we propose to change the nomenclature of the α1256 hexamer to α121-α565 hexamer to better represent the heterotypic interaction of α121 and α565 protomers.