Mapping and Exploring the Collagen-I Proteostasis Network

Abstract

Collagen-I is the most abundant protein in the human body, yet our understanding of how the endoplasmic reticulum regulates collagen-I proteostasis (folding, quality control, and secretion) remains immature. Of particular importance, interactomic studies to map the collagen-I proteostasis network have never been performed. Such studies would provide insight into mechanisms of collagen-I folding and misfolding in cells, an area that is particularly important owing to the prominence of the collagen misfolding-related diseases. Here, we overcome key roadblocks to progress in this area by generating stable fibrosarcoma cells that inducibly express properly folded and modified collagen-I strands tagged with distinctive antibody epitopes. Selective immunoprecipitation of collagen-I from these cells integrated with quantitative mass spectrometry-based proteomics permits the first mapping of the collagen-I proteostasis network. Biochemical validation of the resulting map leads to the assignment of numerous new players in collagen-I proteostasis, and the unanticipated discovery of apparent aspartyl-hydroxylation as a new post-translational modification in the N-propeptide of collagen-I. Furthermore, quantitative analyses reveal that Erp29, an abundant endoplasmic reticulum proteostasis machinery component with few known functions, plays a key role in collagen-I retention under ascorbate-deficient conditions. In summary, the work here provides fresh insights into the molecular mechanisms of collagen-I proteostasis, yielding a detailed roadmap for future investigations. Straightforward adaptations of the cellular platform developed will also enable hypothesis-driven, comparative research on the likely distinctive proteostasis mechanisms engaged by normal and disease-causing, misfolding collagen-I variants, potentially motivating new therapeutic strategies for currently incurable collagenopathies.

Figure 1. Expression of Orthogonally Tagged Collagen-I Strands in HT-1080 Cells

(A) Schematic of collagen-I expression constructs. (B) Immunoblotting analysis of inducible HA-collagen-α1(I) or FLAG-collagen-α2(I) levels in the lysates and media of HT-1080 cells expressing either construct. (C) Immunoblotting analysis of inducible HA-collagen-α1(I) and FLAG-collagen-α2(I) levels in the lysate and media of HT-1080^Col-I cells expressing both constructs. (D) qPCR analysis of unfolded protein response-regulated genes in HT-1080^Col-I cells upon induction of collagen-I expression. Tunicamycin (Tm; 5 μg/mL, 12 h)- and thapsigargin (Tg; 1 μM, 12 h)-mediated unfolded protein response activation are shown as positive controls. qPCR data are reported relative to untreated HT-1080 cells as the mean ±95% confidence interval. (E) Confocal microscopy imaging of collagen-I trafficking in HT-1080^Col-I cells under ER-retention conditions. PDI is an ER marker, LAMP1 is an early lysosome marker, GM130 is a Golgi marker, and DRAQ5 is a nuclear marker. (F) Representative autoradiograms of [³⁵S]-labeled HA-collagen-α1(I) immunoisolated from HT-1080^Col-I media and lysates following induction of collagen-I expression. Control media and lysate were harvested from uninduced HT-1080^Col-I cells. (G) Quantification of autoradiograms in Figure 1F. Collagen-I % remaining was calculated by normalizing the secreted and lysate collagen-I signals at the stated times to the total amount of labeled collagen-I observed at time = 0 h. Collagen-I % secreted was calculated by normalizing the secreted collagen-I signal to the total amount of collagen-I present at time = 0 h. Error bars represent SEM from biological replicates (n = 3).

Figure 2. Molecular Properties of Collagen-I Produced by HT-1080^Col-I Cells

(A) Collagen-α2(I) co-IPs with collagen-α1(I) and vice versa, demonstrating their intracellular association. The control sample represents HT-1080 cells that do not express HA/FLAG-tagged collagen-I upon induction. (B) Trypsin and chymotrypsin digests of collagen-I secreted from primary fibroblasts and HT-1080^Col-I cells demonstrate the presence of a stable, protease-resistant triple helix. See also Figure S1A for complete immunoblots. (C) MS1 scans showing the enrichment of the hydroxylated peptide in the heavy (ascorbate-treated) sample, and enhanced abundance of the unmodified peptide in the medium (ascorbate-deficient) sample. (D) MS1 scans showing that Xaa-position Pro residues 986 in collagen-α1(I) and 707 in collagen-α2(I) (numbering beginning at the first Gly-Xaa-Yaa repeat) are hydroxylated in HT-1080^Col-I cells. See Figure S1B for MS2 scans. (E) Analysis of the N-glycosylation of intracellular HA-tagged collagen-α1(I) immunoprecipitates. In the absence of PNGase-F treatment, HA-antibody reactivity overlaps with ConA reactivity, while treatment with PNGase-F eliminates ConA reactivity. The control sample represents HT-1080 cells that do not express HA-tagged collagen-I upon induction.

Figure 3. Covalent Crosslinking to Enable Robust Co-Immunoprecipitation of the Collagen-I Proteostasis Network

(A) LC-MS/MS-mediated analysis of the stable collagen-I interactome in the absence of covalent crosslinker. The control sample represents HT-1080 cells that do not express HA-tagged collagen-I upon induction. (B) Structure of the covalent crosslinker employed, dithiobis(succinimidyl propionate) (DSP). (C) Optimization of crosslinking conditions for robust co-IP of the collagen-I interactome using HA-antibody beads. The control sample represents HT-1080 cells that do not express HA-tagged collagen-I upon induction. See also Figure S2.

Figure 4. Quantitative, Mass Spectrometry-Based Mapping of the Collagen-I Proteostasis Network

(A) Schematic representation of mass spectrometry workflow employed. (B) Interactomics map of the collagen-I proteostasis network (illustrated by solid black lines) and respective complex components known to interact with each other (illustrated by dotted black lines). (C) Pie chart showing enrichment of secretory pathway proteins in the collagen-I interactome.

Figure 5. Validation and Characterization of Putative Collagen-I Proteostasis Network Components

(A) HA antibody-mediated IP of HA-collagen-α1(I) from HT-1080^Col-I cells coimmunoprecipitates numerous novel or poorly characterized collagen-I interactions identified by mass spectrometry. The control sample represents HT-1080 cells that do not express HA-tagged collagen-I upon induction. (B) Validation of the biological relevance of the interactions identified in the HT-1080 cells by immunoprecipitating HA-tagged wild type collagen-α1(I) from osteosarcoma Saos-2 cells and then immunoblotting. Collagen-I expression was induced by treatment with 1 μg/mL dox and 50 μM ascorbate. The control sample represents untreated Saos-2 cells. (C) Secreted collagen-I levels analyzed by immunoblot in the context of a panel of shRNA stable knockdowns of the indicated proteins in Saos-2 cells. See also Figure S3. (D) MS1 and MS2 spectra with respective peaks assigned for a peptide whose fragmentation pattern is consistent with aspartyl-hydroxylation at residues Asp71 and Asp72 (numbering beginning at collagen-I’s start codon) in the N-propeptide of collagen-α1(I) immunoisolated from HT-1080^Col-I cells. (E) MS2 spectrum with respective peaks labeled that is consistent with aspartyl-hydroxylation at residue Asp71 of collagen-α1(I) immunoisolated from Saos-2 media. For both (D) and (E), asterisks (*) indicate the site of a hydroxylated aspartate or alkylated cysteine. b-Ions are fragments of the parent ion, originating at the N-terminus and extending the number of amino acids into the peptide, as indicated. y-Ions are the same, but originating at the C-terminus of the peptide.

Figure 6. A Role for Erp29 in Collagen-I Proteostasis

(A) Scatter plot of the log₂ SILAC ratios for proteins identified in two or more biological replicates with heavy (ascorbate-treated):light (control) and medium (ascorbate-deficient):light (control) ratios. (B) Scatter plot of the log₂ SILAC ratios for the Disulfide Redox and HSP40/70/90 functional groups showing that these collagen-I interactors are quantitatively enriched under ER-retention (ascorbate-deficient) conditions. (C) Collagen-I secretion from Saos-2 cells under ER-retention (ascorbate-deficient) conditions is increased by Erp29 knockdown, while intracellular collagen-I levels are correspondingly reduced. (D) Model illustrating the role of Erp29 in collagen-I retention/quality control under ascorbate-deficient conditions.