Panoply™ Human GPNMB Over-expressing Stable Cell Line

Xenophagy plays a crucial role in inhibiting intracellular bacterial growth in macrophages. However, the regulatory mechanisms governing autophagosome-lysosome fusion during bacterial infection remain incompletely understood. Here, researchers utilized leprosy as an ideal model to explore the interplay between host defense mechanisms and bacterial infection. They focused on the glycoprotein non-metastatic melanoma protein B (GPNMB), which is highly expressed in macrophages from patients with lepromatous leprosy (L-Lep) and interferes with xenophagy during bacterial infection. Following infection, GPNMB interacts with autophagosome-localized STX17, leading to reduced N-glycosylation at the N296 site of GPNMB. This modification promotes the degradation of SNAP29, thereby preventing the assembly of the STX17-SNAP29-VAMP8 SNARE complex. Consequently, autophagosome-lysosome fusion is disrupted, leading to inhibition of cellular autophagic flux. GPNMB deficiency impairs the proliferation of multiple intracellular bacteria in addition to Mycobacterium leprae in human macrophages, suggesting a general role for GPNMB in intracellular bacterial infections. Furthermore, Gpnmbfl/fl Lyz2-Cre mice exhibited reduced expansion of Mycobacterium marinum compared to controls. Together, these studies reveal a previously unrecognized role for GPNMB in host antimicrobial defense and provide new insights into its regulatory mechanisms in SNARE complex assembly.

Here, researchers assessed the phosphorylation levels of ULK1 and Beclin 1 in GPNMB-overexpressing HEK293T cells. Overexpression of GPNMB did not affect the phosphorylation levels of ULK1 and Beclin 1, indicating that GPNMB has no effect on autophagosome formation (Figure 1A, B). Confocal microscopy revealed that, following infection with Mycobacterium leprae, GPNMB colocalized with the lysosomal marker lysosomal-associated membrane protein 1 (LAMP1) (Figure 1C), but not with the endoplasmic reticulum marker calreticulin, the Golgi marker Golgi matrix protein 130 (GM130), or the mitochondrial outer membrane translocase 20 (TOMM20) (Figure 1C), suggesting that GPNMB may be involved in autophagosome maturation. GPNMB inhibited the fusion of M. leprae LC3+ vacuoles with LAMP1-labeled lysosomes in sgCtrl THP-1 cells (Figure 1D). Furthermore, in GPNMB-knockout THP-1 cells, treatment with chloroquine (CQ), a drug used to inhibit lysosomal degradation (Figure 1E, F), or bafilomycin A1, a late autophagy inhibitor (Figure 1C-F, H, I), restored the amount of LC3-II. Furthermore, the researchers found that the acidic environment and intracellular activity of lysosomes were unaffected by GPNMB. Treatment with EBSS, rapamycin, or IFN-γ disrupted autophagosome maturation in GPNMB-overexpressing HEK293T cells (Figure 1G). Taken together, these results suggest that GPNMB blocks autophagosome-lysosome fusion (autophagosome maturation) but does not affect LC3-II production.

Figure 1. GPNMB blocks the maturation of autophagosomes. (Yan Z, et al., 2025)