Mannose-receptor-mediated clearance of lysosomal alpha-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 alpha-mannosidase from bovine kidney lysosomes.

Bovine kidney lysosomal alpha-mannosidase is a family 38 alpha-mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-beta-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 alpha-mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.

Purification of bovine lysosomal alpha-mannosidase, characterization of its gene and determination of two mutations that cause alpha-mannosidosis.

Bovine kidney lysosomal alpha-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16 kb and its corresponding cDNA contained an open reading frame of 2997 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjacent to a protein sequence of 949 amino acids that was cleaved into five peptides in the mature enzyme; starting with the peptide derived from the N-terminal part of this precursor, their molecular masses were 35/38 (peptide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 kDa (peptide e). Variation in the degree of N-glycosylation accounts for molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide-linked. A T961-->C transition, resulting in Phe321-->Leu substitution, was identified in the cDNA of alpha-mannosidosis-affected Angus cattle. In affected Galloway cattle, a G662-->A transition that causes Arg221-->His substitution was identified. Phe321 and Arg221 are conserved among the alpha-mannosidase class-2 family, indicating that the substitutions resulted from disease-causing mutations in these breeds.

alpha-Mannosidosis: functional cloning of the lysosomal alpha-mannosidase cDNA and identification of a mutation in two affected siblings.

a-Mannosidosis (MIM 248500) is an autosomal recessive lysosomal storage disorder resulting from deficient activity of lysosomal alpha-mannosidase (LAMAN) (EC 3.2.1.24). The disease is characterized by massive intracellular accumulation of mannose-rich oligosaccharides with resulting mental retardation, hearing loss, immune deficiency and skeletal changes. We report here the purification and characterization of human placenta LAMAN. The enzyme is synthesized as a single-chain precursor which is processed into three glycopeptides of 70, 42 and 15 kDa. The 70 kDa peptide is further partially proteolysed into three more peptides that are joined by disulfide bridges. The laman cDNA sequence was assembled from overlapping fragments obtained by PCR on human fibroblast and human lung cDNA. The deduced amino acid sequence contains a putative signal peptide of 48 amino acids followed by a polypeptide sequence of 962 amino acids. Northern blot analyses revealed a single transcript of approximately 3.5 kb present in all tissues examined but at varying levels. Two affected siblings of Palestinian origin were homozygous for a mutation that causes a His-->Leu replacement at a position which is conserved among class 2 alpha-mannosidases from several species.

Characterization of the carbohydrate moiety of human gamma-glutamyltransferases using lectin-blotting and glycosidase treatment.

The presence of both N- and O-linked carbohydrate was demonstrated on gamma-glutamyltransferase (GT) from human liver and kidney. The N-linked carbohydrate constituted 25-30% of the total molecular mass of the enzymes. O-Glycosylation was detected on both subunits of the liver enzyme, but only on the small subunit of the kidney enzyme. Lectin blot analysis indicated that the glycan chains were of the complex type for both the liver and the kidney GT and lectin blotting may to some extent distinguish the two enzymes.

Nickel and iron in saliva of patients with fixed orthodontic appliances.

Saliva was obtained from patients receiving treatment with fixed orthodontic appliances. One saliva sample was taken without appliances, and another at least 3 weeks after placement. In some patients samples were also taken immediately after insertion of the appliance. Nickel and iron were quantified by electrothermal atomic absorption spectroscopy. There was a large scatter in the results. No statistically significant differences were found either in the concentrations or in absolute masses of nickel or iron in samples taken without appliances and in those obtained when the appliances had been in the mouth for at least 3 weeks. For samples taken immediately after placement of the appliance, there was a significant increase in both concentrations and masses of nickel and iron. It thus seems that there is a high initial release of metals, and the effect diminishes with time.