Chondroadherin fragmentation mediated by the protease HTRA1 distinguishes human intervertebral disc degeneration from normal aging

Abstract

Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.

Keywords: Chondroadherin; Extracellular Matrix; HTRA1; IVD; Intervertebral Disc Degeneration; Protease; Protein Degradation; Protein Turnover; Proteolytic Enzymes.

FIGURE 1.

Comparison of CHAD structure in normal discs with age (A) and disc level in the spine (B). All protein extracts were from organ donors without DDD or scoliosis, and the disc level study used tissue from a 60-year-old donor. Extracts were fractionated using SDS-PAGE, and immunoblotting was performed with an anti-CHAD antiserum. NP, nucleus pulposus; AF, annulus fibrosus. These data were reproduced with several samples; however, for clarity, only three representative samples are shown.

FIGURE 2.

Comparison of CHAD fragmentation in normal and degenerate disc protein extracts. Protein extracts from adult discs (normal, aged 60 years; degenerate, aged 56 years) (A) and juvenile scoliotic discs (normal scoliotic, aged 14 years; degenerate scoliotic, aged 15 years) (B) were fractionated using SDS-PAGE and immunoblotted using an anti-CHAD antiserum. NA, normal adult; DA, degenerate adult; NS, non-degenerate scoliotic; DS, degenerate scoliotic. These data were reproduced with several samples; however, for clarity, only four representative samples are shown. The ratio of fragmented to intact CHAD was evaluated in 15 non-degenerate discs aged 26–60 years old and 14 degenerate discs aged 15–70 years old (C). Protein extracts were fractionated using SDS-PAGE, and band intensity was analyzed on immunoblots probed with an anti-CHAD antiserum using ImageQuant TL software and an LAS4000 image analyzer (p = 0.007). Error bars represent S.D.

FIGURE 3.

Comparison of CHAD fragmentation with degree of disc degeneration. Extracted proteins from the same donor (68 years of age) were fractionated by SDS-PAGE, and immunoblotting was performed with an anti-CHAD antiserum. N, normal; MD, mildly degenerate; SD, severely degenerate. These data were reproduced with several samples; however, for clarity, only one representative sample is shown.

FIGURE 4.

Schematic representation of CHAD structure and location of the cleavage site characteristic of disc degeneration within the third leucine-rich repeat. C indicates the location of the 4 cysteine residues at the N-terminal and C-terminal ends of the CHAD leucine-rich repeat region. The site of the cleavage seen in vitro is marked with an arrow between isoleucine 80 and tyrosine 81.

FIGURE 5.

Comparison of CHAD cleavage sites in degenerate juvenile scoliotic (15 years of age) and adult discs (normal, 60 years of age; degenerate, 56 years of age). Extracted proteins were fractionated by SDS-PAGE, and immunoblotting was performed using an anti-CHAD antiserum and an anti-neoepitope antibody (Anti-Neo). N, normal; DS, degenerate scoliotic; DA, degenerate adult. These data were reproduced with several samples; however, for clarity, only three representative samples are shown.

FIGURE 6.

Protease digests of normal disc tissue. Following digestion, extracted proteins were fractionated by SDS-PAGE and probed using an anti-CHAD antiserum. Digestions were performed with MMP-3, -7, -12, and -13 (A); ADAMTS-4 and -5 (B); and cathepsins L, B, and K (C). −, no-enzyme control.

FIGURE 7.

HTRA1 digestion of normal disc tissue. Following digestion, extracted proteins were fractionated using SDS-PAGE and probed with both an anti-CHAD antiserum and an anti-neoepitope antiserum recognizing the in situ cleavage site (Anti-Neo). +, enzyme-digested; −, no-enzyme control; DA, degenerate adult disc extract.

FIGURE 8.

A, comparison of HTRA1 protein levels in juvenile scoliotic (15 years of age) and adult discs (normal, 60 years of age; degenerate, 56 years of age). Equivalent amounts of extracted proteins were fractionated by SDS-PAGE, and immunoblotting was performed using an anti-HTRA1 antiserum. B, verification of HTRA1-generated aggrecan cleavage in degenerate disc samples from two individuals aged 56 and 68 years with disc tissue from a non-degenerate donor aged 60 years. Extracted proteins were fractionated using SDS-PAGE, and immunoblotting was performed using a neoepitope antiserum directed toward the HTRA1 cleavage site in aggrecan (anti-VQTV³⁵⁶). These data were reproduced with multiple tissue samples; however, for clarity, only three are shown.