Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect.

Kretz, Keith A.; Carson, Geoffrey S.; Morimoto, Satoshi; Kishimoto, Yasuo; Fluharty, Arvan L.; O’Brien, John S.

doi:10.1073/pnas.87.7.2541

Cited by 114 publications

(59 citation statements)

References 24 publications

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“…14,15 Mutation N215H has been identified in a patient described as a 4-year-old child with MLD. 13 Mutation T217I has been found in two siblings with a probably milder MLD phenotype than in patient PV, showing first symptoms at 4.5 and 6 years of age respectively, 16 whilst our patient showed walking difficulties at 2 years and mental retardation at 3.…”

Section: Discussionmentioning

confidence: 99%

“…To date five other MLD-causing sap-B mutations have been identified in the PSAP gene. [13][14][15][17][18][19] All patients carrying these mutations are homozygotes for their particular molecular defect. The low number of MLD-causing PSAP mutations identified to date could also be ascribed to a possible underestimation of the disease at the diagnostic level.…”

Section: Discussionmentioning

confidence: 99%

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An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

et al. 1999

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Sphingolipid activator proteins are small glycoproteins required for the degradation of sphingolipids by specific lysosomal hydrolases. Four of them, called saposins, are encoded by the prosaposin gene, the product of which is proteolytically cleaved into the four mature saposin proteins (saposins A, B, C, D). One of these, saposin B, is necessary in the hydrolysis of sulphatide by arylsulphatase A where it presents the solubilised substrate to the enzyme. As an alternative to arylsulphatase A deficiency, deficiency of saposin B causes metachromatic leukodystrophy. We identified a previously undescribed mutation (N215K) in the prosaposin gene of a patient with metachromatic leukodystrophy but with normal arylsulphatase A activity and elevated sulphatide in urine. The mutation involves a highly conserved amino acidic residue and abolishes the only N-glycosylation site of saposin B.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

et al. 1999

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“…They are known to contribute to the proper protein folding and to preserve biological activity by stabilizing the protein conformation and protecting against proteolytic degradation (11)(12)(13). Furthermore, oligosaccharides are important for correct targeting of lysosomal proteins.…”

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confidence: 99%

Functional Analysis of the Glycosylation of Murine Acid Sphingomyelinase

Newrzella

Stoffel

1996

Journal of Biological Chemistry

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Glycosylation plays a crucial role in glycoprotein stability and its correct folding. Murine acid sphingomyelinase (ASM) is a lysosomal glycoprotein. We studied the functional role of its individual N-linked oligosaccharides needed to maintain enzymatic activity and protein stability. Mutagenized cDNA constructs were heterologously expressed. All six potential N-glycosylation sites were modified. Incomplete glycosylation of the most distant C-terminal site resulted in two isoforms. Oligosaccharides at N-84, N-173, and N-611 were found to be of minor importance for enzymatic activity. The glycosylation defect at N-333 or N-393 reduced the enzymatic activity to 40% and at N-518 to less than 20%. These mutations did not effect the K m value. Glycosylation at N-333 and N-393 mainly contributed to the enzyme stability and prevented degradation at lysosomal acidic pH, whereas the low residual enzymatic activity of mutant ASM deficient in glycosylation at N-518 was caused by protein misfolding. The mutant protein was also prone to proteolysis when trapped in the endoplasmic reticulum/cis-Golgi after brefeldin A application. Insufficiently glycosylated ASM formed a stable complex with BiP, an immunoglobulin heavy chain-binding protein, and thus remained in the endoplasmic reticulum.32 PO 4 labeling revealed that the glycosylation mutants of ASM were phosphorylated predominantly at mannose residues of oligosaccharides linked to N-84, N-333, and N-393.Acid sphingomyelinase (ASM, 1 E.C. 3.1.4.12) is an ubiquitous lysosomal hydrolase that cleaves sphingomyelin to ceramide and phosphocholine. In 1987, Quintern et al.(1) purified human acid sphingomyelinase from urine to homogeneity. Subsequent discovery of the primary structure enabled functional expression in an eucaryotic cell culture system (2).Deficient or reduced ASM activity causes sphingomyelin accumulation known in humans as Niemann-Pick disease sphingolipidosis. Depending on the amount of residual enzymatic activity, the neurovisceral type A or the less severe visceral type B of this autosomal recessive disease is observed (3).Recently, several mutations in the ASM gene of Niemann-Pick disease patients have been reported, leading to an enzyme of no or drastically reduced activity. Transgenic mice with a disrupted ASM gene exhibit a phenotype comparable to that of Niemann-Pick disease, type A, patients. The neurological symptoms of these mice are accompanied by the loss of Purkinje cells as revealed by histological studies (4,5).Besides the catabolic function of this lysosomal enzyme, involvement of ASM in the ceramide-dependent signal transduction pathway has been discussed controversially (6 -9). As a lysosomal hydrolase, ASM undergoes several posttranslational processing and targeting steps. The N-terminal signal sequence directs the primary translation product into the ER. The signal sequence, which has a length of approximately 45 amino acids for human as well as murine ASM according to the von Heijne prediction (10), is cleaved during this process. In the ER, o...

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“…For instance, SAPLIP-deficient Caenorhabditis elegans is overcome by bacteria in culture, [24] because the worm is unable to induce bacterial lysis. In humans, deficiencies in saposins A, B or C lead to Krabbe-like disease, [25] metachromatic leukodystropy [26] and Gaucher-like disease, [27] respectively. SAPLIPs are also important contributors in host interactions.…”

Section: Structures Of Parasite Proteinsmentioning

confidence: 99%

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

et al. 2014

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1Mason et al. Structures of parasite proteinsNew interventions against infectious diseases require a detailed knowledge and understanding of pathogen-host interactions and pathogeneses at the molecular level. The combination of the considerable advances in systems biology research with methods to explore the structural biology of molecules is poised to provide new insights into these areas. Importantly, exploring threedimensional structures of proteins is central to understanding disease processes, and establishing structure-function relationships assists in identification and assessment of new drug and vaccine targets. Frequently, the molecular arsenal deployed by invading pathogens, and in particular parasites, reveals a common theme whereby families of proteins with conserved three-dimensional folds play crucial roles in infectious processes, but individual members of such families show high levels of specialisation which is often achieved through grafting particular structural features onto the shared overall fold. Accordingly, the applicability of predictive methodologies based on the primary structure of proteins or genome annotations is limited, particularly when thorough knowledge of molecular level mechanisms is required. Such instances exemplify the need for experimental threedimensional structures provided by protein crystallography, which remain an essential component of this area of research. In the present article, we review two examples of key protein families recently investigated in our laboratories, which could represent intervention targets in the metabolome or secretome of parasites. IntroductionInfectious diseases caused by eukaryotic pathogens represent a major disease burden to humans world-wide. In the absence of effective preventative approaches and new intervention strategies, this burden is likely to increase further, [1,2] compounded by food and water shortages, economic crises, wars and climate change, particularly in disadvantaged countries. In addition, there is evidence of increasing problems with resistance in pathogens against a broad range of chemotherapeutic agents, compromising the treatment of infectious diseases. [3] Moreover, in spite of major efforts, there are very few examples of success in developing new drugs and vaccines against eukaryotic pathogens, [4,5] indicating that alternative methods are needed at a time of major advances in molecular and computer technologies. In our opinion, the development of new treatments requires a detailed understanding of pathogens, host interactions and the molecular processes of disease. For instance, knowledge of the three-dimensional structures of proteins with pivotal roles in disease, and the probing of their underlying biology are crucial for understanding the pathogenesis. Through establishing the relevant structure-function relationships, one can elucidate how individual proteins function, so that new ways of disrupting relevant pathways can be found, in order to facilitate the identification of new drug and vaccine targets. ...

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Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect.

Abstract: Saposins are small, heat-stable glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Saposins A

Cited by 114 publications

References 24 publications

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

Functional Analysis of the Glycosylation of Murine Acid Sphingomyelinase

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

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