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Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice

Anke Suter1, Vincent Everts2, Alan Boyde3, Sheila J. Jones3, Renate Lüllmann-Rauch4, Dieter Hartmann4, Alison R. Hayman5, Timothy M. Cox5, Martin J. Evans5, Tobias Meister1, Kurt von Figura1 and Paul Saftig1,*

1 Zentrum Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany
2 Department of Cell Biology, AMC, Meibergdreef 15, 1105 AZ Amsterdam, and Department of Periodontology, ACTA, University of Amsterdam The Netherlands
3 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
4 Anatomisches Institut, Christian Albrechts Universität Kiel, 24118 Kiel, Germany
5 Department of Medicine, University of Cambridge, Level 5, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK; Wellcome Trust, Institute of Cancer and Developmental Biology; and Department of Genetics, University of Cambridge CB2 1QR, UK



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  		Fig. 1. (A) Generation of LAP/Acp5 doubly deficient mice. Southern blot with external probes for the targeted LAP locus (Saftig et al., 1997) and the Acp5 locus (Hayman et al., 1996). (B) Determination of lysosomal acid phosphatase activity (Waheed et al., 1985) in enriched lysosomal liver fractions of control, single deficient and doubly deficient mice. Acp5 activity was determined after immunoprecipitation with an anti-uteroferrin antibody. (C) LAP/Acp5 doubly knockout mice display an increased mortality starting at about 8 months of age. The results from LAP and Acp5 single knockout as well as control animals are also shown. (D) Hepatosplenomegaly in LAP/Acp5 deficient animal. The spleen and liver of an extreme case of hepatosplenomegaly in LAP/Acp5 knockout animals is shown. (E) Progressive increase in liver weight in LAP/Acp5-deficient mice. A box blot of control (striped boxes) and LAP/Acp5 deficient liver weights is shown from 1-15 month of age. (F) Progressive increase in spleen weight in LAP/Acp5-deficient mice. A box plot of control (striped boxes) and LAP/Acp5 deficient spleen weights is shown from 1-15 month of age.

 


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  		Fig. 2. Lysosomal storage in LAP/Acp5-deficient liver, spleen and kidney. (A,B) Semithin section of a liver of a 3.5 month old control (A) and LAP/Acp5-deficient (B) mouse. Vacuolated Kupffer cells are indicated by arrows. (C,D) Sections through a control and LAP/Acp5-deficient spleen. Numerous vacuolated macrophages are apparent in the double deficient mouse (arrowheads in D); S, sinus. (E,F) LAMP1 immunohistology of control (E) and LAP/Acp5-deficient (F) kidney, demonstrating an increased immunoreactivity in LAP/Acp5-deficient kidney. (G,H) Lectin staining (sWGA) of control (G) and LAP/Acp5-deficient liver (H); cv, central vein. Note the increased labelling of LAP/Acp5-deficient Kupffer cells. Scale bars: 15 µm in A,B; 25 µm in C,D; 400 µm in E,F; 30 µm in G,H.

 


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  		Fig. 3. Electron microscopy of: (A) storage lysosomes (arrows) in hepatocyte in the vicinity of a bile canaliculus of a 6-month-old LAP/Acp5 deficient mouse (H, hepatocyte; bc, bile canaliculus); and (B) Kupffer cell (K) with storage lysosomes (I, Ito cell). (C) Higher magnification of storage lysosomes in a LAP/Acp5-deficient Kupffer cell. (D) LAP/Acp5-deficient spleen macrophage filled with storage lysosomes. (E) Sinus endothelial cell of a LAP/Acp5-deficient spleen with storage vacuoles (F) LAP/Acp5-deficient kidney fibroblast filled with storage vacuoles. Scale bars: 1.4 µm in A; 3.5 µm in B; 0.8 µm in C; 2.4 µm in D; 3.0 µm in E; 4.5 µm in F.

 


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  		Fig. 4. Bone abnormalities in LAP/Acp5-deficient mice. (A-D) Radiographs of 8-week-old mice: (A) control; (B) Lap–/–; (C) Acp5–/–; and (D) Lap–/–/Acp5–/–. Note the foreshortening of long bones in the LAP/Acp5-deficient mouse. (E-H) Light microscopy of growth plates of 3-month-old mice: (E) control; (F) Lap–/–; (G) Acp5–/– and (H) Lap–/–/Acp5–/–. Note the expansion of the cartilaginous growth plates (egp) in Acp5- and LAP/Acp5-deficient mice with disruption, hyperplasia and hypertrophy of the chondrocytes (ps, primary spongiosa). (I,J) PMMA embedded distal femurs of control and Lap–/–/ Acp5–/– after plasma ashing. Imaged with three detectors, used to give red, green and blue signal components. Colour here codes for direction and slope of the internal surfaces exposed by removing PMMA. Control image field is 4 mm in height. (K,L) Localisation of succinylated wheat germ agglutinin (sWGA)-binding sites in growth plates of (K) control bone and (L) LAP/Acp5 doubly deficient bone. Scale bars: 120 µm in E-H; 50 µm in K,L.

 


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  		Fig. 5. Vacuolisation of phosphatase-deficient osteoclasts shown in TEM of post-osmicated, uranyl and lead stained ultra-thin sections. (A) Intracellular vacuole (arrow) in an osteoclast of a LAP-deficient mouse. Note the content of moderate electron density of the vacuole. (B,C) Vacuoles (arrows) in the cytoplasm of an osteoclast of a Acp5-deficient mouse (B) and in an osteoclast from a doubly deficient mouse (C): the vacuoles have a higher electron density and contain linear features that may represent mineral crystallites. (D) High magnification of typical mineral cystallites containing vacuoles of a doubly deficient osteoclast. Scale bars: 0.5 µm in A-C; 130 nm in D.

 


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  		Fig. 6. (A-C) Immunolocalisation of osteopontin in bones. Light microscopic localisation by silver enhancement. (A) Osteopontin localisation in long bone of a control mouse. Note labelling in the bone matrix and the cement lines. Adjacent to the ruffled border of the osteoclast (OC) a low level of label is apparent. (B,C) Localisation of osteopontin in long bone of a doubly deficient mouse. A high level of labelling (arrows) is seen adjacent to the ruffled border of the osteoclast. (D-G) Electron microscopic localisation of osteopontin in bones. (D) Ruffled border area (RB) of an osteoclast of a control mouse. Gold particles (arrowheads) indicate the presence of osteopontin in the bone matrix at some distance from the ruffled border. (E) Ruffled border area of an osteoclast of a LAP-deficient mouse. Note the presence of a small number of gold particles (arrowheads) adjacent to the ruffled border membrane (B, bone). (F) Ruffled border area of an osteoclast of a LAP/Acp5 doubly deficient mouse. A large number of gold particles (arrowheads) are seen adjacent to the ruffled border membrane. (G) Intracellular vacuole (arrow) in an osteoclast of a doubly deficient mouse. The presence of osteopontin in the vacuole is indicated by a high number of gold particles (arrowheads). Scale bars: 20 µm in A-C; 0.18 µm in D; 0.25 µm in E,F; 0.15 µm in G.

 


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  		Fig. 7. Dephosphorylation of osteopontin by lysosomal acid phosphatases. (A) Western blot of recombinant mouse osteopontin incubated for 2 and 3 hours in the presence of bone extracts from control and LAP/Acp5-deficient animals, and analysed using antibodies to osteopontin. Note that the osteopontin immunoreactive bands have disappeared during incubation with control bone extracts while in LAP/Acp5-deficient bone extracts, the immunoreactivity is preserved. (B) Using anti-phosphoserine antibodies, the lack of dephosphorylation of osteopontin after incubation with LAP/Acp5 bone extracts is evident. The serine-phosphorylated osteopontin has disappeared after incubation with bone extracts from control mice. (C) In the presence of proteinase inhibitors, the immunoreactivity is still lost with control bone extracts, whereas inhibition of phosphatase activities with L-tartrate and molybdate prevented dephosphorylation of osteopontin.

 


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  		Fig. 8. Normal dephosphorylation of arylsulfatase A in LAP/Acp5-deficient fibroblasts. Control (Lap/Acp5+/+) and doubly deficient (Lap/Acp5–/–) mouse embryonic fibroblasts were labelled for 6 hours with [35S] or [32P]arylsulfatase A and subsequently chased with non labelled medium up to 24 hours. The disappearance of the labelled arylsulfatase A is followed and the ratio of [32P]/[35S]arylsulfatase A is calculated indicating normal dephosphorylation of arylsulfatase A.

 

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© The Company of Biologists Ltd 2001