Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states.
To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs.
Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes.
Forty NP tissues were snap-frozen in liquid nitrogen (−196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each).
Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan–collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation.
COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy.
The largest aneural and avascular biological structure connecting vertebral bodies is the intervertebral disc (IVD). IVD pathologies are the most responsible for the global epidemic of chronic low back pain (LBP) [
The research was conducted with the appropriate ethical clearance obtained from the institutional review board of Ganga Medical Centre and Hospitals, Coimbatore, India (IRB approval no., 2019/08/06). Nucleus pulposus (NP) tissues were isolated from IVDs from five different disc phenotypes. Under sterile conditions, fetal spines obtained from specimens following the medical termination of pregnancy were dissected for NP harvesting. This group (fetal disk phenotype [FD]) represented a proliferative and developmental phenotype with high proliferative potential. During an anterior release procedure, scoliotic disk phenotype (SD) was harvested from patients with idiopathic scoliosis. These disks represent an early degeneration phenotype because they are usually subjected to additional mechanical stresses while appearing normal on magnetic resonance imaging (MRI). MRI-normal IVDs obtained from brain-dead voluntary organ donors who had no history of LBP exhibited a healthy (normal) disk phenotype (ND). During surgical intervention, two types of samples were obtained for the degenerate group: one from herniated disk phenotypes (DHs) and the other from degenerated disk phenotypes (DD). Excised NP tissues were washed with phosphate buffer solution and immediately snap-frozen in liquid nitrogen (LN2; −196°C) before proteomic analysis. The demographic and Pfirrmann grades are enlisted in
Total proteins were extracted from cartilaginous NP tissue samples using buffers containing salts and detergents, as well as other protease inhibitors. Under aseptic conditions, 200 mg of tissue was pulverized and homogenized with LN2. RIPA (radio-immunoprecipitation assay) and 2% sodium-deoxycholate (SDS) buffers were used for the sequential extraction of hydrophilic and hydrophobic proteins, which were then quantified, cleaned up, and pre-fractionated on 10% SDS-PAGE (polyacrylamide gel electrophoresis), as described in our previous report [
Tryptic digested peptides were purified and subjected to ESI-LC-MS/MS analysis at a concentration of 1,000 femtomole per injection performed in duplicate. The purified peptides were analyzed using an Orbitrap Velos Pro Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode electrospray ionization using an EASY-spray column (PepMap RSLC, C18, 2 μm, 100 Å, 75 μm×50 cm or 15 cm), as described earlier. Proteome Discoverer ver. 1.4 (Thermo Fisher Scientific) was used to identify proteins from raw data generated by tandem mass spectrometric analysis (.raw/.msf). By searching against the UniProt human database, the in-built SequestHT and Mascot search algorithms were used to match tandem mass spectra to peptide sequences. As reported previously [
By mapping the total proteins expressed in the study group (FD, ND, SD, DD, and DH) against the collagen list retrieved from the Matrisome database (
To better understand the biological roles played by collagen types, ShinyGo (
Immunohistochemistry was used to validate the proteomic evidence of COL14A1. Before embedding dissected samples in paraffin, they were stored in formalin. The experiment used 2–5-m sections from paraffin blocks and the tree-step indirect method. Prior to rinsing with ethanol solution and blocking with 0.1% hydrogen peroxide, the blocks were subjected to antigen retrieval using Tris pH 9.5 and borate pH 8.0 in conjugation. A 1:500 dilution of COL14A1 polyclonal antibody (Thermo Fisher Scientific; #cat. no. PA5-49916) was used. Further sections were developed with DAB and Harris hematoxylin before being scanned at 400
IBM SPSS software ver. 25.0 (IBM Corp., Armonk, NY, USA) was used to compare protein expression levels between sample groups. To understand the normal distribution of sample groups, a normality assessment was performed. In the case of a normal distribution,
This study included 40 IVDs, eight in each group (FD, ND, SD, DD, and DH). Tandem mass spectrometric analyses revealed total proteins of 1,050 in FD, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in DD. Collagens, a major component of the ECM, were selectively screened in this study. Of the 28 major collagens reported in the human body, this study identified 24 major collagens with 34 subtypes (
Major collagen expression varied across groups, with 12 collagens identified in the FD, eight in the ND, 14 in the SD, 16 in the DH, and 11 in the DD groups, respectively (
The majority of the ECM is composed of fibril-forming collagens, and types 1, 2, 3, 5, and 11 were identified in our study (
The samples contained relatively short collagens with interrupted triple helices, as well as FACITs 9, 12, 14, 16, 19, and 20 (
Microfibrillar/beaded filament collagen (collagen type 6: COL6A1, COL6A2, COL6A3), network-forming collagen (collagen type 10: COL10A1), and multiplexing collagen (COL15A1 and COL18A1) were also identified in our study (
ShinyGO was used to characterize the collagen types identified at the biological (
An enriched pathway analysis using Reactome pathway browser ver. 3.7 (
Collagenases including MMP types 1, 2, 3, 8, 9, 10, 13, and 14, and cysteine proteases, such as cathepsin K, were identified. Although their basal expression results in normal collagen turnover, their excessive abundance can cause ECM degradation [
COL14A1, which was found to be abundant in fetal disks in this study, was also found to be abundant in bovine fetal disks in a previous study [
Collagen degradation causes ECM disorganization and NP apoptosis, hastening the degenerative process [
We compared the collagen expression of fetal disks with that of healthy adult and degenerate disks to identify collagens with anabolic potential. We also compared healthy adult disks with degenerate disks to identify molecules essential for disk homeostasis and to establish biomarkers for disk degeneration. The precise biological roles of the differentially expressed collagens and the identified potential molecular targets are discussed comprehensively.
Multiplexin (COL15A1), one of the most underreported collagens in humans, was the only collagen found to be specific to fetal disks, representing their role in IVD development. However, there is increasing evidence of its role in numerous physiological and pathological processes apart from maintaining the structural integrity of the ECM [
Several studies have demonstrated the importance of COL-2 in maintaining healthy NP homeostasis [
Another observation in the young developmental phenotypes was the higher expression of COL-6, 9, 11, 12, and 14. Similar to our study, Wu et al. [
Caldeira et al. [
When analyzing the degenerate disk phenotype, a striking observation was the presence of COL-18A1 only in the DH and DD groups. It has been reported as a normal constituent in the basement membrane of eyes [
Our study is unique in that we have documented the collagen expression of 40 IVDs across five different biological phenotypes in humans, which has never been done before. A comparative analysis between developmental, adult, and degenerate phenotypes has revealed novel biomarkers for degeneration and has also identified potential molecular targets for novel therapies in DDD. The proteomic finding was also validated using immunohistochemistry but was done only for COL-14. Our study has provided deep insights into the molecular understanding of IVD in health and disease.
Our study used high-end proteomic technology for the first time to analyze the human IVD across five different biological states, revealing novel molecular targets for therapy and potentially changing the spectrum of care in LBP and DDD. LBP affects 80% of the population at least once in their lifetime, with a global age-adjusted point prevalence of 7.5% [
The results of the study show the importance of collagens other than COL-1 and COL-2 in determining the health and disease of human IVDs. COL-6, 9, 11, 12, and 14 were identified to have higher expression in developmental phenotypes and are, therefore, of regenerative potential in addition to COL-15A1, which was uniquely present in fetal disks. Added to the downregulation of COL-2, the higher expressions of COL-18A1 and COL-10A1 were identified as novel biomarkers of DDD.
This is the 2021 APSS-Asian Spine Journal Best Paper Award.
No potential conflict of interest relevant to this article was reported.
SR, DCR, CT, and MR conceived and formulated the project. SMN and CT contributed to the design of the analysis; performed lab experiments and bulk of data analysis; DCR and SMN wrote and prepared the manuscript. All authors have read through and given the final approval of the submitted publication.
The project was funded by Ganga Orthopaedic Research & Education Foundation (GOREF 2021-09).
We acknowledge the efforts of Ms M. Sujitha and Ms M. Dhanalakshmi for assistance in LC–MS/MS experiments.
Collagen frequency detected in samples.
Bar graph representation of identified various collagen types across study groups.
Functional analysis of collagens.
Bar plot representing the expression of procollagens, collagenases, and inhibitors across study groups (fetal [FD], normal [ND], scoliotic [SD], herniated [DH], and degenerated [DD] disk phenotype).
Validation of COL14A1 by immunohistochemistry. Ten fetal samples and five normal disk phenotype samples were used. Seven of fetal samples showed strong cytoplasmic and extracellular matrix (ECM) staining. In contrast, only two adult samples showed mild staining in ECM (magnification ×400, scale bar=50 μm). Positive samples are stained brown.
Demographic details of the study subjects considered under this study
Study group with the mean age of the subjects | Age | Sex | Pfirrmann grade |
---|---|---|---|
FD (wk) | 24 | 1 | |
24 | 1 | ||
24 | 1 | ||
24 | 1 | ||
24 | 1 | ||
24 | 1 | ||
24 | 1 | ||
24 | 1 | ||
ND (45.88±17.05 yr) | 57 | F | 3 |
43 | M | 2 | |
22 | M | 2 | |
55 | F | 2 | |
67 | M | 2 | |
63 | M | 2 | |
28 | M | 1 | |
32 | F | 2 | |
SD (21±6.63 yr) | 17 | F | 2 |
29 | M | 2 | |
29 | M | 2 | |
29 | M | 2 | |
16 | F | 2 | |
16 | F | 2 | |
16 | F | 2 | |
16 | F | 2 | |
DH (44.75±15.84 yr) | 32 | M | 4 |
70 | F | 5 | |
38 | F | 4 | |
37 | M | 4 | |
43 | M | 4 | |
45 | M | 3 | |
67 | F | 3 | |
26 | M | 4 | |
DD (58.5±12.39 yr) | 60 | F | 3 |
61 | F | 4 | |
42 | M | 5 | |
64 | F | 5 | |
49 | M | 4 | |
78 | M | 5 | |
69 | F | 4 | |
45 | F | 4 |
Values are presented as mean±standard deviation or number.
FD, fetal disk phenotype; ND, normal disk phenotype; SD, scoliotic disk phenotype; DH, herniated disk phenotype; DD, degenerated disk phenotype; F, female; M, male.
Comparison of various types of collagen reported in the literature and identified in our study population
Collagen subfamily | Reported major types of collagen | Reported variable α chains | Localization | Collagens identified in our study |
---|---|---|---|---|
Fibril-forming | Collagen type I | α1(I), α2(I) | Skin, bone, dentin, cementum, tendons, ligaments | COL1A1, COL1A2 |
Fibril-forming | Collagen type II | α1(II) | Vitreous humor, intervertebral disc | COL2A1 |
Fibril-forming | Collagen type III | α1(III) | Skin, blood vessels, lymphoid tissues | COL3A1 |
Basement membrane | Collagen type IV | α1(IV), α2(IV), α3(IV), α4(IV), α5(IV), α6(IV) | Basal laminae | COL4A1 |
Fibril-forming | Collagen type V | α1(V), α2(V), α3(V), α4(V) | Basal laminae, peridontal tissues, dermis | COL5A1, COL5A2, COL5A3 |
Micro-fibrillar/beaded filament | Collagen type VI | α1(VI), α2(VI), α3(VI), α4(VI), α5(VI), α6(V) | Ligament, placenta, skin, cartilage | COL6A1, COL6A2, COL6A3, COL6A5, COL6A6 |
Anchoring | Collagen type VII | α1(VII) | Skin and mucosa | COL7A1 |
Network-forming | Collagen type VIII | α1(VIII) | Cornea and endothelium | COL8A1, COL8A2 |
FACITs | Collagen type IX | α1(IX), α2(IX), α3(IX) | Cartilage, vitreous humor | COL9A1, COL9A2 |
Network-forming | Collagen type X | α1(X) | Hypertrophic chondrocytes in the proliferative zone of fetal cartilage | COL10A1 |
Fibril-forming | Collagen type XI | α1(XI), α2(XI), α3(XI) | Articular cartilage, testis, trachea, tendons, trabecular bone, skeletal muscle, placenta, lung, and the neoepithelium of the brain | COL11A1, COL11A2 |
FACITs | Collagen type XII | α1(XII) | Bone, ligaments, fibrocartilage, smooth muscle and skin | COL12A1 |
MACIT | Collagen type XIII | α1(XIII) | Epidermis, hair follicle | - |
FACITs | Collagen type XIV | α1(XIV) | Connective tissues | COL14A1 |
Multiplexin | Collagen type XV | α1(XV) | Connective tissue stroma | COL15A1 |
FACITs | Collagen type XVI | α1(XVI) | Dermal fibroblasts and keratinocytes | COL16A1 |
MACIT | Collagen type XVII | α1(XVII) | Epithelial hemidesmosome of skin and mucosa | - |
Multiplexin | Collagen type XVIII | α1(XVIII) | Oral mucosa | COL18A1 |
FACITs | Collagen type XIX | α1(XIX) | Breast, colon, kidney, liver, placenta, prostate, skin | COL19A1 |
FACITs | Collagen type XX | α1(XX) | Biased expression in brain and testis | COL20A1 |
FACITs | Collagen type XXI | α1(XXI) | Skeletal muscle, pancreas, lymph node | COL21A1 |
FACITs | Collagen type XXII | α1(XXII) | Tissue junctions of muscles, tendons, the heart, articular cartilage, and skin | COL22A1 |
MACIT | Collagen type XXIII | α1(XXIII) | Skeletal and heart muscle | - |
Fibril-forming | Collagen type XXIV | α1(XXIV) | Bone cornea | COL24A1 |
Membrane-bound brain specific | Collagen type XXV | α1(XXV) | Neurons | COL25A1 |
Fibril-forming | Collagen type XXVI | α1(XXVI) | Adult testis and ovary | COL26A1 |
Fibril-forming | Collagen type XXVII | α1(XXVII) | Cartilage, eye, ear, lungs | COL27A1 |
Multiplexin | Collagen type XXVIII | α1(XXVIII) | Neuronal tissue | - |
(−) indicates the absence of collagen type in our study. Col6A5 has been designated as COL29A1.
FACIT, fibril associated collagen with interrupted helices; MACIT, membrane associated collagen with interrupted helices.
Top 34 significant pathways identified for observed collagen types in this study using Reactome pathway browser ver. 3.7 sorted based on
Pathway name | Protein involved | Found | FDR | |
---|---|---|---|---|
Assembly of collagen fibrils and other multimeric structures | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL9A1;COL10A1;COL6A3 | 15/67 | 1.11E-16 | 1.44E-15 |
Collagen formation | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL20A1;COL9A1;COL10A1;COL6A3 | 18/104 | 1.11E-16 | 1.44E-15 |
Collagen biosynthesis and modifying enzymes | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL20A1;COL9A1;COL10A1;COL6A3 | 18/76 | 1.11E-16 | 1.44E-15 |
Collagen degradation | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL9A1;COL10A1;COL6A3 | 17/69 | 1.11E-16 | 1.44E-15 |
Collagen chain trimerization | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL20A1;COL9A1;COL10A1;COL6A3 | 18/44 | 1.11E-16 | 1.44E-15 |
ECM organization | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL20A1;COL9A1;COL10A1;COL6A3 | 18/329 | 1.11E-16 | 1.44E-15 |
Degradation of the ECM | COL18A1;COL15A1;COL14A1;COL11A1;COL12A1;COL11A2;COL19A1;COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL9A1;COL10A1;COL6A3 | 17/148 | 1.11E-16 | 1.44E-15 |
Integrin cell surface interactions | COL1A1;COL18A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL9A1;COL10A1;COL6A3;COL19A1 | 11/86 | 3.22E-13 | 3.47E-12 |
ECM proteoglycans | COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 8/79 | 3.47E-13 | 3.47E-12 |
NCAM1 interactions | COL2A1;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 6/44 | 8.19E-11 | 7.37E-10 |
MET activates PTK2 signaling | COL1A1;COL2A1;COL1A2;COL5A1;COL11A1;COL11A2 | 6/32 | 1.82E-09 | 1.46E-08 |
Non-integrin membrane-ECM interactions | COL1A1;COL2A1;COL1A2;COL5A1;COL11A1;COL11A2;COL10A1 | 7/61 | 4.87E-09 | 3.41E-08 |
MET promotes cell motility | COL1A1;COL2A1;COL1A2;COL5A1;COL11A1;COL11A2 | 6/45 | 1.23E-08 | 8.61E-08 |
NCAM signaling for neurite out-growth | COL2A1;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 6/70 | 1.90E-07 | 1.14E-06 |
Signaling by PDGF | COL2A1;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 6/70 | 1.50E-06 | 8.99E-06 |
Signaling by MET | COL1A1;COL2A1;COL1A2;COL5A1;COL11A1;COL11A2 | 6/88 | 6.87E-06 | 3.43E-05 |
Anchoring fibril formation | COL1A1;COL1A2 | 2/15 | 1.10E-04 | 5.50E-04 |
Signaling by receptor tyrosine kinases | COL1A1;COL2A1;COL1A2;COL5A1;COL6A2;COL11A1;COL6A1;COL11A2;COL9A1;COL6A3 | 10/593 | 2.02E-04 | 0.001 |
Crosslinking of collagen fibrils | COL1A1;COL1A2 | 2/24 | 2.80E-04 | 0.001 |
Syndecan interactions | COL1A1;COL1A2;COL5A1 | 3/29 | 0.001 | 0.004 |
Scavenging by class A receptors | COL1A1;COL1A2 | 2/48 | 0.001 | 0.004 |
Platelet adhesion to exposed collagen | COL1A1;COL1A2 | 2/16 | 0.005 | 0.024 |
GP1b-IX-V activation signalling | COL1A1;COL1A2 | 2/12 | 0.010 | 0.040 |
Axon guidance | COL2A1;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 6/584 | 0.018 | 0.056 |
Binding and uptake of ligands by scavenger receptors | COL1A1;COL1A2 | 2/167 | 0.022 | 0.066 |
GPVI-mediated activation cascade | COL1A1;COL1A2 | 2/43 | 0.026 | 0.078 |
Nervous system development | COL2A1;COL5A1;COL6A2;COL6A1;COL9A1;COL6A3 | 6/620 | 0.026 | 0.079 |
Immunoregulatory interactions between a lymphoid and a non-lymphoid cell | COL1A1;COL2A1;COL1A2 | 3/316 | 0.029 | 0.086 |
RUNX2 regulates osteoblast differentiation | COL1A1 | 2/34 | 0.031 | 0.085 |
Laminin interactions | COL18A1 | 1/31 | 0.033 | 0.087 |
RUNX2 regulates bone development | COL1A1 | 2/43 | 0.040 | 0.087 |
Fibronectin matrix formation | COL1A1 | 1/7 | 0.042 | 0.087 |
Interleukin-4 and interleukin-13 signaling | COL1A2 | 2/211 | 0.050 | 0.098 |
FDR, fractional degradation rate; ECM, extracellular matrix; NCAM, neural cell adhesion molecule; MET, mesenchymal–epithelial transition; PTK2, protein tyrosine kinase 2; PDGF, platelet-derived growth factor; GP, glycoprotein; RUNX2, Runt-related transcription factor 2.