The severe pediatric disorder Mucolipidosis II (ML-II) is characterizedby multiple developmental defects including pronounced skeletal,craniofacial, and cartilage abnormalities. ML-II patients have mutations inthe gene encoding the catalytic activity of the enzyme GlcNAc-1-phosphotransferase leading to impaired biosynthesis of mannose-6-phosphate (Man-6-P), the key recognition marker for sorting of lysosomalhydrolases to lysosomes. Though the genetic bases for this disorder arenow well established, the molecular mechanisms underlying thepathophysiology of individuals with ML-II remain poorly understood. In aneffort to investigate the developmental defects of this disease we analyzedthe expression level and activity of a number of lysosomal hydrolasesusing a zebrafish model for ML-II. The present work addresses the role oflysosomal and matrix proteases in the cartilage pathogenesis of ML-II.Several cathepsins and matrix metalloproteinases (MMPs) were found to bepresent at elevated levels in ML-II. Analysis of cathepsin K, an enzymeinvolved in bone and cartilage homeostasis, revealed the enzyme is tightlyregulated during early cartilage development in zebrafish. Further studiesshowed cathepsin K is present at normal levels but subject to enhancedproteolytic activation in ML-II. The pharmacological or genetic suppressionof cathepsin K was shown to alleviate several aspects of the craniofacialdefects as well as the elevated activities of other proteases. Cathepsin Kwas found to be a highly Man-6-P modified enzyme capable of functioningat a pH consistent with the extracellular space, and deficient within sortedchondrocytes. By contrast, the related enzyme cathepsin D was poorlyMan-6-P modified and present at normal levels in chondrocytes,suggesting cathepsin-specific hypersecretion occurs in zebrafish. Removalof specific N-glycans of cathepsin K and exogenous expression in wildtypeembryos revealed that the enzyme is subject to enhanced activationupon loss of Man-6-P modification and is capable of generating cartilagephenotypes analogous to those seen in ML-II. Further studies using a felinemodel revealed that increased MMP activity is a general feature of ML-II,and is accompanied by the increased expression of proinflammatorycytokines. Collectively, these results indicate that the inappropriateextracellular activity of cathepsin K plays a central role in the cartilagepathogenesis of ML-II.