Infantile malignant osteopetrosis (IMO) is a fatal bone disorder disease caused primarily by TCIRG1 mutations that impair osteoclast (OC)-mediated bone resorption. Although TCIRG1 deficiency is known to disrupt lysosomal acidification through defective V‑ATPase function, the pathogenic mechanisms by which the TCIRG1 variant (c.1371delC) regulates TCIRG1 stability, trafficking, and assembly, remain poorly defined. This study aimed to clarify the molecular mechanism of TCIRG1 dysfunction and identify its critical regulatory partners to advance the understanding of IMO pathogenesis.

We established a set of comprehensive in vitro OC models, including TCIRG1-knockout, TCIRG1-overexpressing, and TCIRG1-variant (c.1371delC) OCs derived from RAW264.7 cells. OC differentiation, membrane potential dynamics, lysosomal acidification, autophagic flux, bone‑resorptive capacity, and inflammatory cytokine secretion were systematically assessed. A tamoxifen-inducible IMO mouse model and human TCIRG1 (c.1371delC) knockin rescue lines, was used to evaluate in vivo phenotypes. Lysosome‑enriched proteomics, co‑immunoprecipitation (co-IP), bioinformatics, and molecular docking were employed to identify and validate TCIRG1‑interacting proteins, while protein structural modeling assessed how the TCIRG1-variant impacts V-ATPase conformation and binding stability.

TCIRG1 loss or the c.1371delC mutation impaired OC differentiation and bone resorption, disrupted lysosomal acidification and autophagic flux, and altered cytokine profiles. IMO mice exhibited growth retardation, systemic osteosclerosis, diminished movement, and shortened survival. Lysosomal-enriched proteomics and co-IP analysis identified VPS33B was the key TCIRG1‑associated regulator. Additionally, we proved that VPS33B directly interacted with TCIRG1, was necessary for TCIRG1 lysosomal localization, and supported V-ATPase activity and lysosome-endosome fusion. Meanwhile, VPS33B loss recapitulated key features of TCIRG1 deficiency, which could not be rescued by TCIRG1 (c.1371delC). The protein structural modeling analysis revealed that the c.1371delC frameshift disrupted a transmembrane helix domain in TCIRG1, weakening electrostatic and hydrophobic interactions with VPS33B and destabilizing the TCIRG1-VPS33B complex. 

Overall, this study identifies VPS33B as a previously unrecognized upstream regulator of TCIRG1 that essential for maintaining lysosomal homeostasis and V‑ATPase function in osteoclasts. The VPS33B-TCIRG1 axis is a central determinant of OC dysfunction in IMO and represents a promising target for therapeutic intervention.