Excessive activity of cathepsin K is associated with cartilage defects in a zebrafish model of mucolipidosis II

Abstract

SUMMARYThe severe pediatric disorder mucolipidosis II (ML-II; also known as I-cell disease) is caused by defects in mannose 6-phosphate (Man-6-P) biosynthesis. Patients with ML-II exhibit multiple developmental defects, including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model of ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP) transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S) and MMP-13 during early development. Unlike MMP-13, for which higher levels of protein were detected, the sustained activity of cathepsin K at later stages seemed to result from its abnormal processing and activation. Inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.

Site-1 protease deficiency causes human skeletal dysplasia due to defective inter-organelle protein trafficking

“…In contrast, the S1P patient has increased levels of urine N-telopeptides, suggesting abnormal ECM degradation due to abnormally secreted lysosomal enzymes with collagenase activity in the absence of S1P. Thus, these data indicate that the defective S1P-GPT-lysosome axis plays an additional contribution to the skeletal dysplasia in this patient ( Figure 6F) (27).…”

The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase is a substrate recognition module

“…GlcNAc-1-phosphotransferase activity was determined as described earlier, using αMM as substrate and UDP-[ 3 H]GlcNAc as donor. Cathepsin K activity assays were performed using fluorogenic substrates, as previously described (15). CI-MPR affinity chromatography was performed as described previously (14).…”

“…Embryos depleted of GlcNAc-1-phosphotransferase exhibit decreased mannose phosphorylation of lysosomal acid hydrolases, craniofacial and cardiac defects, and altered development of pectoral fins and otic vesicles. The level of active cathepsin K is strikingly elevated at 3 d postfertilization (dpf) because of abnormal processing and activation (15). This excess cathepsin K activity appears to have a central role in the pathogenesis of the morphologic cartilage defects.…”

“…This excess cathepsin K activity appears to have a central role in the pathogenesis of the morphologic cartilage defects. All of these abnormalities are prevented by the coinjection of WT gnptab mRNA with the morpholino (15). This system provided an opportunity to test the consequences of the K732N mutation in a developing embryo.…”

Analysis of Mucolipidosis II/III GNPTAB Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme GlcNAc-1-phosphotransferase Involved in Catalytic Function and Lysosomal Enzyme Recognition

“…6A). MLII zebrafish typically exhibit 1) short flattened jaws with Meckel's (M) cartilages that do not protrude beyond the eyes, 2) decreased distance between the Meckel's and ceratohyal (CH) structures, 3) a straightened angle of articulation between the ceratohyal elements, and 4) reduced Alcian blue staining of posterior structures (17,20). Therefore, these criteria (outlined in Fig.…”