amamutdb.no: A relational database for MAN2B1 allelic variants that compiles genotypes, clinical phenotypes, and biochemical and structural data of mutant MAN2B1 in α-mannosidosis.

α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by mutations in the MAN2B1 gene, encoding lysosomal α-mannosidase. The disorder is characterized by a range of clinical phenotypes of which the major manifestations are mental impairment, hearing impairment, skeletal changes, and immunodeficiency. Here, we report an α-mannosidosis mutation database, amamutdb.no, which has been constructed as a publicly accessible online resource for recording and analyzing MAN2B1 variants (http://amamutdb.no). Our aim has been to offer structured and relational information on MAN2B1 mutations and genotypes along with associated clinical phenotypes. Classifying missense mutations, as pathogenic or benign, is a challenge. Therefore, they have been given special attention as we have compiled all available data that relate to their biochemical, functional, and structural properties. The α-mannosidosis mutation database is comprehensive and relational in the sense that information can be retrieved and compiled across datasets; hence, it will facilitate diagnostics and increase our understanding of the clinical and molecular aspects of α-mannosidosis. We believe that the amamutdb.no structure and architecture will be applicable for the development of databases for any monogenic disorder.

Identification of 83 novel alpha-mannosidosis-associated sequence variants: functional analysis of MAN2B1 missense mutations.

The lysosomal storage disorder alpha-mannosidosis is caused by deficiency of the enzyme lysosomal alpha-mannosidase (MAN2B1). In this study, 96 disease-associated sequence variants were identified in 130 unrelated alpha-mannosidosis patients from 30 countries. Eighty-three novel variants were detected, extending the mutation spectrum from 42 to 125. In total, 256 of the 260 mutant alleles (98.5%) were identified. Most of the variants were unique to each family, however, c.2248C>T (p.Arg750Trp) was detected in 50 patients from 16 countries, and accounted for 27.3% of the disease alleles. Haplotype analysis revealed that the c.2248T variant was present on four MAN2B1 haplotype backgrounds, where one major haplotype accounted for 95% of the alleles. The distribution of the c.2248T-associated haplotypes differed remarkably from those of the control populations, suggesting that c.2248C>T has occurred on a few ancestral haplotypes where the major haplotype subsequently has spread by founder effects. The disease-associated missense mutations were introduced into the human MAN2B1 cDNA, expressed in cell culture and assayed for MAN2B1 activity. The majority of the variants were inactive, however, ten showed MAN2B1 activity above background, and more detailed studies are necessary to further evaluate the pathogenicity of these variants.

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function.

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

The transcriptional regulation and cell-specific expression of the MAPK-activated protein kinase MK5.

The mitogen-activated protein kinase (MAPK) cascades regulate important cellular processes, including growth, differentiation, apoptosis, embryogenesis, motility and gene expression. Although MAPKs mostly appear to be constitutively expressed, the transcript levels of some MAPK-encoding genes increase upon treatment with specific stimuli. This applies to the MAPK-activated protein kinases MK2 and MK3. By contrast, the transcriptional regulation of the related MK5 has not yet been studied. The MK5 promoters of mouse, rat and human contain a plethora of putative transcription factor sites, and the spatio-temporal expression of MK5 suggests inducible transcription of the gene. We examined the transcription pattern of MK5 in different tissues, and studied the kinetics of MK5 expression at the transcriptional and/or translation level in PC12 cells exposed to arsenite, forskolin, KCl, lipopolysaccharide, spermine NONOate, retinoic acid, serum, phorbol ester, temperature shock, and vanadate. Cells exposed to forskolin display a transient increase in MK5 mRNA, despite their unaltered MK5 protein levels. The MK5 promoters of human, mouse and rat contain a cAMP-responsive element that binds the cAMP-responsive element-binding protein (CREB) in vitro. Luciferase reporter constructs containing an 850-base pair human MK5 promoter fragment encompassing the CRE showed a basal activity that was 10-fold higher than the corresponding construct in which the CRE motif was deleted. siRNA-mediated depletion of CREB had no effect on the endogenous MK5 protein levels. Several binding motifs for heat shock factor are dispersed in the mouse and rat promoter, and temperature shock transiently enhanced the MK5 transcript levels. None of the other tested stimuli had an effect on the MK5 mRNA or protein levels. Our results indicate an inducible regulation of MK5 transcription in response to specific stimuli. However, the MK5 protein levels remained unaffected by all the stimuli tested. There is still no explanation for the discrepancy between the increased mRNA and unchanged MK5 protein levels.

The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription.

SPBP (Stromelysin-1 PDGF responsive element binding protein) is a ubiquitously expressed 220 kDa nuclear protein shown to enhance or repress the transcriptional activity of various transcription factors. A yeast two-hybrid screen, with the extended plant homeodomain (ePHD) of SPBP as bait, identified TopBP1 (topoisomerase II beta-binding protein 1) as a candidate interaction partner of SPBP. TopBP1 has eight BRCA1 carboxy-terminal (BRCT) domains and is involved in DNA replication, DNA damage responses and in the regulation of gene expression. The interaction between SPBP and TopBP1 was confirmed in vitro and in vivo, and was found to be mediated by the ePHD domain of SPBP and the BRCT6 domain of TopBP1. Both SPBP and TopBP1 enhanced the transcriptional activity of Ets1 on the c-myc P1P2- and matrix metalloproteinase-3 (MMP3) promoters. Together they displayed a more than additive effect. Both proteins were associated with these promoters. The involvement of TopBP1 was dependent on the serine 1159 phosphorylation site, known to be important for transcriptional activation. Depletion of endogenous SPBP by siRNA treatment reduced MMP3 secretion by 50% in phorbol ester-stimulated human fibroblasts. Taken together, our results show that TopBP1 and SPBP interact physically and functionally to co-operate as co-activators of Ets1.

Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.

Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).

Alpha-mannosidosis: correlation between phenotype, genotype and mutant MAN2B1 subcellular localisation.

BACKGROUND: Alpha-mannosidosis is caused by mutations in MAN2B1, leading to loss of lysosomal alpha-mannosidase activity. Symptoms include intellectual disabilities, hearing impairment, motor function disturbances, facial coarsening and musculoskeletal abnormalities. METHODS: To study the genotype-phenotype relationship for alpha-mannosidosis 66 patients were included. Based on the predicted effect of the mutations and the subcellular localisation of mutant MAN2B1 in cultured cells, the patients were divided into three subgroups. Clinical and biochemical data were collected. Correlation analyses between each of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed to describe the genotype-phenotype correlations. The phenotype parameters were modelled by the mutation group and age as a covariate. P values of <0.05 were considered as statistically significant. RESULTS: Complete MAN2B1 genotypes were established for all patients. We found significantly higher scores in the Leiter-R test, lower concentrations of CSF-oligosaccharides, higher point scores in the Bruininks-Oseretsky Test of Motor Proficiency subtests (BOT-2); Upper limb coordination and Balance, and a higher FVC% in patients in subgroup 3, harbouring at least one variant that allows localisation of the mutant MAN2B1 protein to the lysosomes compared to subgrou 2 and/or subgroup 1 with no lysosomal localization of the mutant MAN2B1 protein. CONCLUSION: Our results indicate a correlation between the MAN2B1 genotypes and the cognitive function, upper limb coordination, balance, FVC% and the storage of oligosaccharides in CSF. This correlation depends on the subcellular localisation of the mutant MAN2B1 protein.

Alpha-mannosidosis and mutational analysis in a Turkish patient.

We present a case of alpha-mannosidosis with its mutational analysis. She was referred to our hospital with the provisional diagnosis of mucolipidosis. She was the first child of second-degree relative parents. She had a coarse face with flat and wide nasal bridge, hepatosplenomegaly, umbilical hernia, lumbar gibbus, motor and mental retardation and deafness. On peripheral blood smear, lymphocytes revealed vacuoles and neutrophils contained some granules resembling Reilly bodies seen in mucopolysaccharidosis (MPS). Based on these findings, the diagnosis of alpha-mannosidosis was suspected. Her urine oligosaccharide chromatography showed an abnormal pattern with a heavy trisaccharide band. Enzyme studies on white cells confirmed a deficiency of alpha-mannosidase activity, which was 2.6 micromol/g/hr. Her DNA analysis showed a S453Y mutation.

Sisters with alpha-mannosidosis and systemic lupus erythematosus.

UNLABELLED: Alpha-mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). Here, we report two sisters with alpha-mannosidosis who developed systemic lupus erythematosus (SLE). The sisters were both homozygous for a one bp deletion within the LAMAN gene resulting in a truncated gene product. The coincidence of alpha-mannosidosis and SLE are discussed with regard to both clinical and molecular findings. CONCLUSION: alpha-mannnosidosis may contribute to the onset of systemic lupus erythematosus in predisposed patients.