Thomas Braulke

Missense mutations in N-acetylglucosamine-1-phosphotransferase α/β subunit gene in a patient with mucolipidosis III and a mild clinical phenotype

Mucolipidosis type III (ML III, pseudo‐Hurler polydystrophy), an autosomal recessive inherited disorder of lysosomal enzyme targeting is due to a defective N‐acetylglucosamine 1‐phosphotransferase (phosphotransferase) activity and leads to the impaired formation of mannose 6‐phosphate markers in soluble lysosomal enzymes followed by their increased excretion into the serum. Mutations in the phosphotransferase γ subunit gene (GNPTAG) have been reported to be responsible for ML III. Here we report on a 14‐year‐old adolescent with a mild clinical phenotype of ML III. He presented with progressive joint stiffness and swelling. Urinary oligosaccharide and mucopolysaccharide excretion was normal. Lysosomal enzyme activities were significantly elevated in the serum and decreased in cultured fibroblasts. Impaired trafficking of the lysosomal protease cathepsin D (CtsD) was confirmed by metabolic labeling of the patients fibroblasts. Neither mutations in the GNPTAG gene nor alterations in the GNPTAG mRNA level were detected whereas the steady state concentration of the 97 kDa GNPTAG dimer was reduced. Most importantly, the patient is homozygous for a pathogenic nucleotide substitution and a polymorphism in the phosphotransferase α/β subunit gene (GNPTA). The data indicate that defects in genes other than GNPTAG can be linked to ML III contributing to the variability of the phenotype.

C-Terminal Prenylation of the CLN3 Membrane Glycoprotein Is Required for Efficient Endosomal Sorting to Lysosomes

Mutations in the polytopic lysosomal membrane glycoprotein CLN3 result in a severe neurodegenerative disorder. Previous studies identified two cytosolic signal structures contributing to lysosomal targeting. We now examined the role of glycosylation and the C‐terminal CAAX motif in lysosomal transport of CLN3 in non‐neuronal and neuronal cells. Mutational analysis revealed that in COS7 cells, CLN3 is glycosylated at asparagine residues 71 and 85. Both partially and non‐glycosylated CLN3 were transported correctly to lysosomes. Mevalonate incorporation and farnesyltransferase inhibitor studies indicate that CLN3 is prenylated most likely at cysteine 435. Substitution of cysteine 435 reduced the steady‐state level of CLN3 in lysosomes most likely because of impaired sorting in early endosomal structures, particularly in neuronal cells. Additionally, the cell surface expression of CLN3 was increased in the presence of farnesyltransferase inhibitors. Alteration of the spacing between the transmembrane domain and the CAAX motif or the substitution of the entire C‐terminal domain of CLN3 with cytoplasmic tails of mannose 6‐phosphate receptors have demonstrated the importance of the C‐terminal domain of proper length and composition for exit of the endoplasmic reticulum. The data suggest that co‐operative signal structures in different cytoplasmic domains of CLN3 are required for efficient sorting and for transport to the lysosome.

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.

Increased expression of lysosomal acid phosphatase in CLN3‐defective cells and mouse brain tissue

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by defective function of the lysosomal membrane glycoprotein CLN3. The activity of the lysosomal acid phosphatase (LAP/ACP2) was found to be significantly increased in the cerebellum and brain stem of Cln3‐targeted mice during the early stages of postnatal life. Histochemical localization studies revealed an increased LAP/ACP2 staining intensity in neurons of the cerebral cortex of 48‐week‐old Cln3‐targeted mice as compared with controls. Additionally, the expression of another lysosomal membrane protein LAMP‐2 was increased in all brain areas. Knockdown of CLN3 expression in HeLa cells by RNA interference also resulted in increased LAP/ACP2 and LAMP‐2 expression. Finally in fibroblasts of two juvenile neuronal ceroid lipofuscinosis patients elevated levels of LAP/ACP2 were found. Both activation of gene transcription and increased protein half‐life appear to contribute to increased LAP/ACP2 protein expression in CLN3‐deficient cells. The data suggest that lysosomal dysfunction and accumulation of storage material require increased biogenesis of LAP/ACP2 and LAMP‐2 positive membranes which makes LAP/ACP2 suitable as biomarker of Batten disease.

Regulation of the components of the 150 kDa IGF binding protein complex in cocultures of rat hepatocytes and Kupffer Cells by 3′,5′-cyclic adenosine monophosphate

In the circulation, most of IGFs are bound to a high molecular mass complex of 150 kDa that consists of IGF‐I (or IGF‐II), IGFBP‐3 and the acid‐labile subunit (ALS). Within rat liver, biosynthesis of these components has been localized to different cell populations with hepatocytes as source of ALS and nonparenchymal cells (endothelial and Kupffer cells (KC)) as source of IGFBP‐3. In the present study, the regulatory effects of the cAMP analogs dibutyryl‐cAMP (db‐cAMP) and 8‐bromo‐cAMP (8‐br‐cAMP) on IGF‐I, ALS, and IGFBP expression were evaluated in primary cultures of rat hepatocytes, KC as well as in cocultures of hepatocytes and KC. In cocultures, biosynthesis of IGFBP‐3 and ALS was inhibited dose‐dependently by db‐cAMP and 8‐br‐cAMP while that of IGF‐I, IGFBP‐1, and ‐4 was stimulated as demonstrated by ligand and Northern blotting. IGFBP‐3 expression in primary cultures of pure KC did not respond to cAMP treatment indicating the importance of a cellular interaction between KC and hepatocytes for the decreased IGFBP‐3 synthesis. The inhibition of IGFBP‐3 in db‐cAMP‐treated cocultures was due to a decrease of IGFBP‐3 mRNA level accompanied by a reduced cellular degradation of IGFBP‐3. We conclude that cAMP stimulate the biosynthesis of IGF‐I, IGFBP‐1, and ‐4 in cocultures of hepatocytes and KC thereby enabling the formation of binary IGF/IGFBP complexes while the formation of the 150 kDa complex is impaired through downregulation of IGFBP‐3 and ALS. This complex regulation may be a prerequisite for the effects of cAMP‐dependent hormones on the transfer of IGFs from circulation to peripheral tissues.

Pathogenic mutations cause rapid degradation of lysosomal storage disease-related membrane protein CLN6.

One variant form of late infantile neuronal ceroid lipofuscinosis is an autosomal recessive inherited neurodegenerative lysosomal storage disorder caused by mutations in the CLN6gene. The function of the polytopic CLN6 membrane protein localized in the endoplasmic reticulum is unknown. Here we report on expression studies of three mutations (c.368G>A, c.460‐462delATC, c.316insC) found in CLN6 patients predicted to affect transmembrane domain 3 (p.Gly123Asp), cytoplasmic loop 2 (p.Ile154del) or result in a truncated membrane protein (p.Arg106ProfsX26), respectively. The rate of synthesis and the stability of the mutant CLN6 proteins are reduced in a mutation‐dependent manner. None of the mutations prevented the dimerization of the CLN6 polypeptides. The particularly rapid degradation of the p.Arg106ProfsX26 mutant which is identical with the mutation in the murine orthologue Cln6 gene in the nclf mouse model of the disease, can be strongly inhibited by proteasomal and partially by lysosomal protease inhibitors. Both degradative pathways seem to be sufficient to prevent the accumulation/aggregation of the mutant CLN6 polypeptides in the endoplasmic reticulum.

Alteration of the insulin‐like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin‐like growth factors I and II (IGF‐I, IGF‐II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low‐density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF‐I and ‐II; (ii) synthesized low levels of IGFBP‐2, ‐3, ‐4, and ‐5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF‐I and ‐II expressions increased continuously in parallel with an upregulation of IGFBP‐2, ‐3, ‐4, and ‐5 whereas the IGF‐I receptor and IGF‐II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF‐IR, but low concentrations of IGFBP‐2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF‐IR expression, but increase of mitoinhibitory IGFBP‐2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells. J. Cell. Biochem. 102: 28–40, 2007.

Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions

M6P-dependent transport routes are essential for B cell functions in vivo and humoral immunity in mouse and human.

Impaired bone remodeling and its correction by combination therapy in a mouse model of mucopolysaccharidosis-I

Mucopolysaccharidosis-I (MPS-I) is a lysosomal storage disease (LSD) caused by inactivating mutations of IDUA, encoding the glycosaminoglycan-degrading enzyme α-l-iduronidase. Although MPS-I is associated with skeletal abnormalities, the impact of IDUA deficiency on bone remodeling is poorly defined. Here we report that Idua-deficient mice progressively develop a high bone mass phenotype with pathological lysosomal storage in cells of the osteoblast lineage. Histomorphometric quantification identified shortening of bone-forming units and reduced osteoclast numbers per bone surface. This phenotype was not transferable into wild-type mice by bone marrow transplantation (BMT). In contrast, the high bone mass phenotype of Idua-deficient mice was prevented by BMT from wild-type donors. At the cellular level, BMT did not only normalize defects of Idua-deficient osteoblasts and osteocytes but additionally caused increased osteoclastogenesis. Based on clinical observations in an individual with MPS-I, previously subjected to BMT and enzyme replacement therapy (ERT), we treated Idua-deficient mice accordingly and found that combining both treatments normalized all histomorphometric parameters of bone remodeling. Our results demonstrate that BMT and ERT profoundly affect skeletal remodeling of Idua-deficient mice, thereby suggesting that individuals with MPS-I should be monitored for their bone remodeling status, before and after treatment, to avoid long-term skeletal complications.