Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.

Indium-111-labelled white blood cells ((111)In-WBCs) are currently considered the tracer of choice in the diagnostic work-up of suspected active chronic osteomyelitis (COM). Previous studies in a limited number of patients, performed with dedicated PET systems, have shown that [(18)F]2'-deoxy-2-fluoro- D-glucose (FDG) imaging may offer at least similar diagnostic accuracy. The aim of this prospective study was to compare FDG imaging with a dual-head coincidence camera (DHCC) and (111)In-WBC imaging in patients with suspected COM. Thirty consecutive non-diabetic patients with possible COM underwent combined skeletal scintigraphy (30/30 patients), (111)In-WBC imaging (28/30 patients) and FDG-PET with a DHCC (30/30 patients). During diagnostic work-up, COM was proven in 11/36 regions of suspected skeletal infection and subsequently excluded in 25/36 regions. In addition, soft tissue infection was present in five patients and septic arthritis in three. (111)In-WBC imaging in 28 patients was true positive in 2/11 regions with proven COM and true negative in 21/23 regions without further evidence of COM. False-positive results occurred in two regions and false-negative results in nine regions suspected for COM. Most of the false-negative results (7/9) occurred in the central skeleton. If the analysis was restricted to the 18 regions with available histology ( n=17) or culture ( n=1), (111)In-WBC imaging was true positive in 2/18 regions, true negative in 8/18 regions, false negative in 7/18 regions and false positive in 1/18 regions. FDG-DHCC imaging was true positive in 11/11 regions with proven COM and true negative in 23/25 regions without further evidence of COM. False-positive results occurred in two regions. If the analysis was restricted to the 19 regions with available histology ( n=18) or culture ( n=1), FDG-DHCC imaging was true positive in 9/9 regions with proven COM and true negative in 10/10 regions without further evidence of COM. It is concluded that FDG-DHCC imaging is superior to (111)In-WBC scintigraphy in the diagnosis of COM in the central skeleton and therefore should be considered the method of choice for this indication. This seems to hold true for peripheral lesions as well, but in our series the number of cases with proven infection was too small to permit a final conclusion.

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser 475 -Glu 272 -Asp 360 catalytic triad, and an octahedrally coordinated Ca 2؉ -binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp 276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed.