ST6GALNAC4 Promotes Diffuse Large B-Cell Lymphoma Progression Via Mediating Sialylation of Bmpr-1B at Thr24

Sialylation is a terminal modification of cell glycosylation, including α-2,3-, α-2,6-, and α-2,8-forms, which exert a dominant role in cell recognition and immune regulation. Among them, α-2,6-sialylation and α-2,6-sialyltransferases have shown promising potential in the study of tumor mechanisms and treatment, but their function remains unknown in diffuse large B-cell lymphoma (DLBCL). This study aims to explore the function and underlying mechanism of α-2,6-sialylation in DLBCL.

We first detected the expression of α-2,6-sialylation in lymph node tissues from DLBCL patients and reactive hyperplasia cases with informed consent. DLBCL tissues exhibited higher levels of α-2,6-sialylation compared to the control cases. Survival analysis revealed that DLBCL patients with elevated α-2,6-sialylation levels showed poorer clinical outcomes (P=0.0003). Within the sialyltransferase family, elevated expression of the α-2,6-sialyltransferase ST6GALNAC4 was found to be associated with a poor prognosis in DLBCL patients (P<0.05).

The above findings prompted us to investigate the biological function of ST6GALNAC4 in DLBCL. Knockdown of ST6GALNAC4 declined cell proliferation and induced cell cycle arrest in the G0/G1 phase. Conversely, overexpression of the gene facilitated cell proliferation. Moreover, the oncogenic role of ST6GALNAC4 was confirmed in DLBCL xenograft models. Both ST6GALNAC4 deficiency and sialyltransferase inhibitor-treated groups exhibited significantly delayed tumor growth compared to the control group.

To explore the underlying mechanism of ST6GALNAC4 in DLBCL tumorigenesis, we performed mass spectrometry to identify interacting proteins of ST6GALNAC4. Since sialic acids are typically found at glycan termini on the cell surface, we primarily focused on cell membrane proteins as potential targets of ST6GALNAC4. Among them, BMPR-1B showed a significant positive correlation with ST6GALNAC4 (P<0.001). The correlation was also validated in DLBCL tissues. BMPR-1B, a member of the TGF-β superfamily, is implicated in tumorigenesis and progression. Co-immunoprecipitation confirmed the interaction between ST6GALNAC4 and BMPR-1B, and ST6GALNAC4 knockdown significantly reduced the protein expression of BMPR-1B in DLBCL cell lines. Notably, knockdown of ST6GALNAC4 led to an enrichment of the SMAD signaling pathway, as indicated by KEGG analysis. Furthermore, in vitro and in vivo experiments verified that inhibition of BMPR-1B could inhibit cell growth. Therefore, BMPR-1B was identified as a downstream target of ST6GALNAC4.

We further elucidated the molecular mechanism by which ST6GALNAC4 regulates the expression of BMPR-1B through sialylation. Lectin immunoprecipitation demonstrated that ST6GALNAC4 mediated the sialylation of BMPR-1B. A followed cycloheximide chase experiment was carried out to show that ST6GALNAC4 knockdown decreased the half-life of BMPR-1B protein. We then used site mutations to determine the important modification sites on BMPR-1B. The potential site of BMPR-1B sialylation was predicted by the NetOGlyc web, where Thr24, Thr109, and Ser20 displayed potent potential to be sialylated. We mutated these sites respectively and examined the sialylation level, and found that both Thr109 and Thr24 mutations dramatically reduced the sialylation level of BMPR-1B. Importantly, ST6GALNAC4 significantly enhanced the sialylation levels of wild-type and Thr109-mutant BMPR-1B, but not Thr24-mutant BMPR-1B. This suggested that ST6GALNAC4 specifically mediated sialylation at Thr24. We then confirmed that the Thr24 mutation affected BMPR-1B half-life compared to wild-type BMPR-1B. Furthermore, we validated that ST6GALNAC4 knockdown decreased levels of pSMAD1/5/8, while overexpression of ST6GALNAC4 had the opposite effect. Importantly, overexpression of BMPR-1B could partially reverse the effects of ST6GALNAC4, showing that the oncogenic effects of ST6GALNAC4 were mediated by BMPR-1B. Taken together, these findings suggest that ST6GALNAC4 mediates BMPR-1B sialylation at Thr24, affecting its protein stability and ultimately regulating the SMAD signaling pathway in DLBCL.

In conclusion, our results revealed that ST6GALNAC4 induced α-2,6-sialylation of BMPR-1B, which promoted malignant progression of DLBCL. This underscores the potential of ST6GALNAC4 as a novel prognostic biomarker and therapeutic target for DLBCL.