Despite the significance of glycoproteins for extracellular matrix assembly in cartilage tissue, little is known about the regulation of the chondrocyte glycophenotype under inflammatory conditions. The present study aimed to assess the effect of IL-1β and TNF-α on specific features of the glycophenotype of primary human chondrocytes in vitro. Using LC-MS, we found that both cytokines increased overall sialylation of N- and O-glycans and induced a shift towards α-(2→3)-linked sialic acid residues in chondrocyte glycoproteins. These results were supported by quantitative PCR showing increased expression of α-(2→3) sialyltransferases in treated cells. Moreover, we found that both IL-1β and TNF-α induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans. In contrast, core α- (1→6)-fucosylation of chondrocyte N-glycans was found to be reduced particularly by TNF-α. In summary, inflammatory conditions induce specific alterations of the chondrocyte glycophenotype which might affect cell-matrix interactions or the function of endogenous lectins.

Objective: Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either α-2,3 or α-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans. Methods: The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1β (IL-1β) or tumour necrosis factor-α (TNF-α) on primary cells were assessed. Results: Primary human chondrocytes predominantly express α-2,6-specific SiaTs and accordingly, α-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of α-2,3-linked sialyl residues and, correspondingly, reduced levels of α-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards α-2,3-linked sialic acids and α-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1β or tumour necrosis factor-alpha (TNF-α). Conclusion: The expression of the differentiated chondrocyte phenotype is linked to the ratio of α-2,6- to α-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.