Ole K. Tollersrud

Spectrum of Mutations in α-Mannosidosis

alpha-Mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). The resulting intracellular accumulation of mannose-containing oligosaccharides leads to mental retardation, hearing impairment, skeletal changes, and immunodeficiency. Recently, we reported the first alpha-mannosidosis-causing mutation affecting two Palestinian siblings. In the present study 21 novel mutations and four polymorphic amino acid positions were identified by the screening of 43 patients, from 39 families, mainly of European origin. Disease-causing mutations were identified in 72% of the alleles and included eight splicing, six missense, and three nonsense mutations, as well as two small insertions and two small deletions. In addition, Southern blot analysis indicated rearrangements in some alleles. Most mutations were private or occurred in two or three families, except for a missense mutation resulting in an R750W substitution. This mutation was found in 13 patients, from different European countries, and accounted for 21% of the disease alleles. Although there were clinical variations among the patients, no significant LAMAN activity could be detected in any of the fibroblast cultures. In addition, no correlation between the types of mutations and the clinical manifestations was evident.

Mannose-receptor-mediated clearance of lysosomal α-mannosidase in scavenger endothelium of cod endocardium

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua). Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.

Identification of Asp197 as the catalytic nucleophile in the family 38 α-mannosidase from bovine kidney lysosomes

Bovine kidney lysosomal α‐mannosidase is a family 38 α‐mannosidase involved in the degradation of glycoproteins. The mechanism‐based reagent, 5‐fluoro‐β‐L‐gulosyl fluoride, was used to trap a glycosyl–enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis. Taking into consideration sequence alignments of this region with those of other α‐mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.

Molecular heterogeneity for bovine α-mannosidosis: PCR based assays for detection of breed-specific mutations

DNA tests, based on the polymerase chain reaction (PCR), were developed for the detection of two breed-specific mutations responsible for the autosomal recessive disorder bovine alpha-mannosidosis. The tests involve separate amplification of two exons of the lysosomal alpha-mannosidase gene followed by restriction enzyme digestion of the amplicons. We demonstrate that one of the mutations, the 662G-->A transition, is responsible for alpha-mannosidosis in Galloway cattle. The other mutation, the 961T-->C transition, is uniquely associated with alpha-mannosidosis in Angus, Murray Grey and Brangus cattle from Australia. The 961T-->C mutation was also detected in Red Angus cattle exported from Canada to Australia as embryos. All 39 animals classified as heterozygotes on the basis of biochemical assays were heterozygous for one of the two mutations. None of 102 animals classified as homozygous-normal on the basis of biochemical assays possessed the mutations. Our results indicate that the two breed-specific mutations may have arisen in Scotland and by the export of animals and germplasm disseminated to America, New Zealand and Australia.

Characterization of a novel α-mannosidosis-causing mutation and its use in leukocyte genotyping after bone marrow transplantation

Abstractα-Mannosidosis is a lysosomal storage disorder caused by deficiency of lysosomal α-mannosidase (LAMAN). Major symptoms include mental retardation, skeletal changes and recurrent infections. Recently, a successful bone marrow transplantation (BMT) in an α-mannosidosis patient was reported. Here we show that this patient was homozygous for a novel mutation, a 1-bp insertion (1197–1198insA) in exon 9 of the LAMAN gene. By using this mutation as a marker, we demonstrate that 1 year post-BMT, the LAMAN genotype of the patient’s leukocytes was identical to that of the donor. This method of genotyping blood cells is a fast and accurate way to monitor the colonization of donor bone marrow cells.

Leucoencéphalopathie de l’adulte associée à un déficit en alpha-mannosidase

Resume L’Alpha-mannosidose (MIM248500) est une maladie de transmission autosomique recessive due a un deficit en alpha-mannosidase, enzyme necessaire a la degradation de nombreux oligosaccharides et glycoproteines. On distingue deux types. Le type 1 ou forme infantile et un type 2 ou forme juvenile/adulte. Nous rapportons le cas d’un homme de 51 ans ayant des troubles de la marche evoluant depuis l’âge de 40 ans associe a une leucoencephalopathie due a un deficit en alpha-mannosidase.