Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1

Abstract

Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with β-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. Conclusions— Our results provide the first evidence that CathA acts in vivo as an endothelin-1–inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.

Neuraminidase-1, a Subunit of the Cell Surface Elastin Receptor, Desialylates and Functionally Inactivates Adjacent Receptors Interacting with the Mitogenic Growth Factors PDGF-BB and IGF-2

“…27 Because increased proteolytic activity of cell-surface-delivered PPCA in transfected cells could potentially inactivate other mitogenic polypeptides (eg, endothelin-1 or angiotensin II), 83,84 results obtained from such an experimental model might not be conclusive. However, we demonstrated that treatment of cultured human AoSMCs with blocking antibody raised to human Neu 1 22 or with the competitive sialidase inhibitor ddNANA, which inhibits the activity of Neu1, 27 induced a significant up-regulation in their proliferation in response to fetal bovine serum.…”

Regulation of Phagocytosis in Macrophages by Neuraminidase 1

“…In particular, the components of the multienzyme complex, Neu1, CathA, and ␤-galactosidase (or its alternatively spliced elastin-binding form), participate in processing of endothelin-1 (21,33), assembly of the elastic fibers (21,34,35), pro-inflammatory response in macrophages (36), migration, invasion, and adhesion of cancer cells (37), proliferation of aortic smooth muscle cells (38), and exocytosis (39). In humans, genetic defects in CathA cause disruption of the complex and trigger galactosialidosis (MIM 256540), a severe multisystemic disease characterized by combined deficiency of Neu1, ␤-galactosidase, and CathA (for review, see Ref.…”

“…Animals-CathA S190A mice carrying the point c.571AGCϾGCA (S190A) mutation in the CathA gene and CathA S190A-Neo mice carrying, in addition, a PGK-Neo cassette in intron 7 were generated through the targeted disruption of the CathA gene as described (21). The CathA S190A-Neo and CathA S190A mice ranging from 4 to 12 weeks of age were compared with the appropriate wild-type (WT) littermate controls.…”

“…To determine protein loading, the blot was stained with Ponceau S (Sigma). (21). In the first strain (CathA S190A ) we replaced the nucleophil of the CathA active site, Ser 190 , with Ala, which abolished the enzymatic activity, whereas the mutant protein retained its ability to activate Neu1.…”

“…To clarify the physiological role of Neu1 and CathA in the antigen-presenting immune cells, we have compared essential functional properties of macrophages (MØ) and immature dendritic cells (DC) from the genetically targeted mice having either a single CathA or a double CathA/Neu1 deficiency (21). The obtained data suggest that the cell surface Neu1 activates the phagocytosis in MØ and DC presumably through desialylation of multiple receptors, including Fc receptors for immunoglobulin G (Fc␥R).…”