CTSA Trafficking in Hepatocytes Is Governed by RalA GTPase Activity

The study demonstrates that cathepsin A (CTSA) co-localizes with the lysosomal marker LAMP1 in AML12 mouse hepatocytes, yielding a Pearson’s correlation coefficient of r = 0.818. Immunoprecipitation assays reveal that HA-CTSA binds preferentially to FLAG-RalA pre-loaded with the non-hydrolyzable GTP analogue GTP?S, indicating interaction with the active form of RalA. In contrast, GDP-loaded FLAG-RalA shows markedly weaker binding. Loss of the RalA-activating subunit RalGAPB (RalGAPB-KO) diminishes CTSA-lysosomal colocalization and increases the proportion of CTSA detected in culture supernatants, suggesting that RalA activity promotes lysosomal targeting of CTSA while restraining its secretion.

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Experiments employed AML12 cells transfected with HA-CTSA, HA-Tmem192, or pLEX-HA lentiviruses, and C57BL/6J mice maintained on either normal chow diet (NCD) or high-cholesterol diet (HCD) for up to 20 weeks. RalGAPB-KO AML12 cells and mice with hepatocyte-specific CTSA deletion (sgCTSA) served as loss-of-function models. Control groups included wild-type (WT) cells and sgLacZ-treated mice. All assays were performed with biological replicates ranging from n = 2 to n = 12, as detailed below.

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• CTSA processing: In liver tissue after 4 weeks on NCD or HCD, the CTSA-lyso/CTSA-pre ratio differed significantly between genotypes (exact values not provided).

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• Plasma CTSA: After 20 weeks on HCD, plasma CTSA levels were elevated in RalGAPB-KO mice (n = 12).

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• Secretion assay: CTSA in supernatants of WT versus RalGAPB-KO AML12 cells showed a higher supernatant-to-lysate ratio in the knockout (n = 2–3).

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• LDLR surface expression: Non-permeabilized AML12 cells displayed reduced plasma-membrane LDLR fluorescence intensity in RalGAPB-KO and CTSA-deficient conditions (n = 32–47 cells per group).

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• LDL uptake: DiI-LDL internalization was lower in RalGAPB-KO and CTSA-deleted cells (n = 58–100 cells).

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• Gene expression: Hepatic Ldlr mRNA levels were decreased in sgCTSA mice (n = 7) relative to sgLacZ controls (n = 9).

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• Metabolic phenotypes: sgCTSA mice exhibited altered body weight, liver-to-body weight ratio, plasma glucose, and plasma triglycerides compared with sgLacZ controls (sample sizes 7–9).

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• Lysosomal integrity: Primary hepatocytes from sgCTSA mice showed reduced LysoTracker-positive puncta and lower fluorescence intensity (n = 15 cells).

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The data link impaired CTSA lysosomal trafficking to diminished LDL receptor abundance at the plasma membrane and reduced LDL particle uptake. Correspondingly, CTSA deletion correlates with altered systemic lipid parameters, including plasma triglyceride levels, suggesting that CTSA activity influences hepatic cholesterol handling.

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By establishing RalA as a regulator of CTSA intracellular routing, the work identifies a mechanistic axis that connects GTPase signaling to lysosomal enzyme distribution and downstream LDLR-mediated lipid uptake. Disruption of this axis—through RalGAPB loss or CTSA deficiency—produces measurable changes in hepatic lipid metabolism in mouse models.

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The investigation is confined to murine hepatocyte cultures and C57BL/6J mice; translational relevance to human liver physiology remains to be determined. Further studies are needed to elucidate how modulation of the RalA-CTSA pathway might be leveraged for therapeutic control of dyslipidemia.

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