Identification of N-Glycoproteins of Knee Cartilage from Adult Osteoarthritis and Kashin-Beck Disease Based on Quantitative Glycoproteomics, Compared with Normal Control Cartilage

Abstract

Glycoproteins are involved in the development of many diseases, while the type and content of N-glycoproteins in the cartilage of osteoarthritis (OA) and Kashin–Beck disease (KBD) are still unclear. This research aims to identify N-glycoproteins in knee cartilage patients with OA and KBD compared with normal control (N) adults. The cartilage samples were collected from gender- and age-matched OA (n = 9), KBD (n = 9) patients, and N (n = 9) adults. Glycoproteomics and label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) obtained N-glycoproteins of KBD and OA. A total of 594 N-glycoproteins and 1146 N-glycosylation peptides were identified. The identified data were further compared and analyzed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein–Protein Interactions (PPI). Pairwise comparison of the glycoproteins detected in the three groups showed that integrin beta-1 (ITGB1), collagen alpha-1 (II) chain (COL2A1), collagen alpha-1 (VII) chain (COL7A1), carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 4 (CHST-4), thrombospondin 2 (THBS2), bone morphogenetic protein 8A (BMP8A), tenascin-C (TNC), lysosome-associated membrane protein (LAMP2), and beta-glucuronidase (GUSB) were significantly differentially expressed. GO results suggested N-glycoproteins mainly belonged to protein metabolic process, single-multicellular and multicellular organism process, cell adhesion, biological adhesion, and multicellular organism development. KEGG and PPI results revealed that key N-glycoproteins were closely related to pathways for OA and KBD, such as phagosome, ECM-receptor interaction, lysosome, focal adhesion, protein digestion, and absorption. These results reflected glycoprotein expression for OA and KBD in the process of ECM degradation, material transport, cell–cell or cell–ECM interaction, and information transduction. These key significantly differentially expressed N-glycoproteins and pathways lead to the degeneration and degradation of the cartilage of OA and KBD mainly by disrupting the synthesis and catabolism of basic components of ECM and chondrocytes and interfering with the transfer of material or information. The key N-glycoproteins or pathways in this research are potential targets for pathological mechanisms and therapies of OA and KBD.

Thrombospondin-2 acts as a critical regulator of cartilage regeneration: A review

“…To further determine the role of TSP-2, exogenous TSP-2 was added to chondroprogenitor cells in microculture and revealed that TSP-2 induced chondrogenic effects on cells. [16,43] A study by Taylor et Table 2 TSP-2-related molecular regulation in chondrogenesis.…”

“…Hankenson KD, et al [41] (2000) The role of TSP-2 in cartilage regeneration and repair hUCB-MSCs/ Cartilage TSP-2 expression is elevated in OA Jeong SY, et al [16] (2013) Han J, et al [43] (2022) Cartilage and bone TSP-2 affects the ratio of bone to cartilage formation…”

“…To further determine the role of TSP-2, exogenous TSP-2 was added to chondroprogenitor cells in microculture and revealed that TSP-2 induced chondrogenic effects on chondroprogenitor cells. [16,43] A study by Taylor et al [44] showed that TSP-2-deficient mice had reduced expression of COL-II and Sox9 (markers of chondrocyte differentiation) but increased expression of osteocalcin (a marker of osteoblast differentiation). Chondrogenesis was restored in TSP-2-deficient mice through the expression of TSP-2 using an adenovirus 3 days after fracture.…”

A New Fluorescent Probe Tool: ERNathG

“…β- d -Glucuronidase (GUS, EC 3.2.1.31) is widely found in mammalian tissues, plants, insects, and microbes, and its sequences and structures from different sources are homologous . Therefore, its detection has not only the specificity of identifying site structure but also the universality of applications related to cancer treatment course and gut bacteria monitoring. − GUS hydrolyzes glucuronide to generate free glucuronic acid and aglycone, the latter of which can be rationally designed and artificially synthesized according to the requirements of different objects to be tested …”

“…Thus, alkalis are commonly adopted to adjust the pH for achieving a complete dissociation of the probes; however, this will cause GUS deactivation, in tandem with a stoppage of the enzymatic reaction, thereby limiting the applications of these probe molecules such as for some continuous monitoring. (2) The probes are usually applied in an aqueous medium, which may cause the probes to tend to diffuse away from the detection site, thus missing the opportunity to detect GUS in situ. Besides, the relatively short Stokes shift and excitation wavelength suffer from interference from excitation leakage during the detection and imaging of GUS.…”