Biomedical subjects

Konrad Sandhoff

Publications and source records attributed to Konrad Sandhoff.

At least 37 records · Page 2Linked to original sources

PubMed2005

Saposin C stimulates growth and invasion, activates p42/44 and SAPK/JNK signaling pathways of MAPK and upregulates uPA/uPAR expression in prostate cancer and stromal cells.

AIM: To determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily. METHODS: We employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells. RESULTS: Saposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies. CONCLUSION: Saposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer.

Cell Division↗

PubMed2005

Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family.

Animals↗

PubMed2004

Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation.

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

Animals↗

PubMed2004

Prosaptide TX14A stimulates growth, migration, and invasion and activates the Raf-MEK-ERK-RSK-Elk-1 signaling pathway in prostate cancer cells.

BACKGROUND: Prosaposin is a neurotrophic factor. Prosaposin knock-out mice have been reported to develop a number of abnormalities, including atrophy of the prostate gland and mitogen-activated protein kinase (MAPK)-inactivation in prostate epithelial cells. These abnormalities underscore a potential fundamental role in prostate development. The trophic factor activity of prosaposin has been localized at a specific amino terminal portion of the molecule that has been the source for a number of biologically active peptides called prosaptides (e.g., TX14A). The expression and function of prosaposin in prostate cancer is not known. METHODS: Using conventional protein expression analysis, immunohistochemical staining, cell proliferation assays, and in vitro invasion assays, we determined the expression of prosaposin and the effect of prosaptide TX14A on cell growth/death protection, motility, invasion, and MAPK signal transduction pathway in prostate cancer cells. RESULTS: We found a higher expression of prosaposin in androgen-independent (AI) prostate cancer cells (PC-3 and DU-145) than in androgen-dependent (AD) LNCaP or normal prostate epithelial cells. Immunohistochemical staining on benign and malignant prostate tissues revealed an intense cytoplasmic anti-prosaposin immunoreactivity in tumor cells, as well as stromal, endothelial, and inflammatory mononuclear cells. The intensity of staining was proportional to the overall Gleason's score. In addition, we demonstrated that TX14A stimulates cell proliferation/survival, migration, and invasion, and activates the Raf-MEK-ERK-RSK-Elk-1 signaling cascade of the MAPK pathway. CONCLUSIONS: These results are suggestive of a potential pleuripotent regulatory function for prosaposin in prostate cancer.

Cell Division↗

PubMed2004

Severe subacute GM2 gangliosidosis caused by an apparently silent HEXA mutation (V324V) that results in aberrant splicing and reduced HEXA mRNA.

We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child.

Alternative Splicing↗

PubMed2004

Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine.

The tricyclic antidepressant desipramine causes a decrease in cellular acid sphingomyelinase (A-SMase, EC 3.1.4.12) activity when added to culture medium of human fibroblasts. This effect can be prevented by incubation of the cells with the protease inhibitor leupeptin, which suggests that desipramine induces proteolytic degradation of the lysosomal enzyme. By using surface plasmon resonance (SPR, Biacore) we were able to monitor the interactions of A-SMase and substrate-containing lipid bilayers immobilized on the surface of a Pioneer trade mark L1 sensor chip. SPR binding curves show that the enzyme hardly dissociates from the lipid surface at acidic pH values. On the other hand, a drop in binding signals (resonance units, RU) of approximately 50% occurred after injection of 20 mM desipramine. Our findings indicate that desipramine interferes with the binding of A-SMase to the lipid bilayers and thereby displaces the enzyme from its membrane-bound substrate. The application of control substances suggests a key role for the cationic moiety of desipramine. We hypothesize that the displacement of the glycoprotein A-SMase from the inner membranes of late endosomes and lysosomes by desipramine renders it susceptible to proteolytic cleavage by lysosomal proteases.

Antidepressive Agents, Tricyclic↗

PubMed2004

Saposin C is required for lipid presentation by human CD1b.

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.

Antigen Presentation↗

PubMed2004

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Chromatography↗

PubMed2004

Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism.

During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.

Acyltransferases↗

PubMed2004

Loss of keratin 10 is accompanied by increased sebocyte proliferation and differentiation.

Here, we present strong evidence that the targeted deletion of keratin 10 (K10) alters sebocyte differentiation in mice, mediated by an increased proliferation and differentiation of cells located in the periphery of the glands. This was not accompanied by the induction of the proliferation-associated keratins K6, K16 and K17. Sebaceous gland cells of K10-/- mice showed an accelerated turnover and secreted more sebum including wax esters, triglycerides, and cholesterol esters. The levels of the major epidermal lipids ceramides and cholesterol were also increased, whereas glycosylceramides and sphingomyelin were decreased which was not based on altered sphingolipid biosynthesis. The amount of Cer(OS), covalently bound to the cornified envelope, remained unchanged, as well as the amount of loricrin and involucrin. In agreement with the unaltered expression of beta-catenin and its targets cyclin D1 and c-Myc, we conclude that the altered composition of the suprabasal intermediate filament cytoskeleton in K10-/- mice increased the differentiation of epidermal stem cells towards the sebocyte lineage.

Animals↗

PubMed2004

Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation.

The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information.

Amino Acid Sequence↗

PubMed2004

Phospholipid synthesis is decreased in neuronal tissue in a mouse model of Sandhoff disease.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes for the beta-subunit of beta-hexosaminidase A and B, resulting in ganglioside GM(2) accumulation in the brain. We now demonstrate that phospholipid metabolism is altered in both cultured neurons and in brain tissue from a mouse model of Sandhoff disease, the Hexb-/- mouse. Metabolic labelling using [methyl-(14)C]choline and l-[3-(3)H]serine demonstrated reduced incorporation of [methyl-(14)C]choline into phospholipids in brain tissue but not in liver or spleen. Phospholipid mass was also reduced in brain. The activities of CTP : phosphocholine cytidylyltransferase (CCT) and phosphatidylserine synthase were also reduced in brain tissue from Hexb-/- mice, probably because of post-translational modification as no changes were observed in levels of enzyme expression. The relevance of these findings to Sandhoff disease in human patients is strengthened by observations made over 30 years ago on autopsy tissue of Tay Sachs and Sandhoff disease patients, in which reduced phospholipid levels were observed. We suggest that changes in phospholipid metabolism are not simply because of loss of neuronal tissue as a result of degeneration but rather may cause degeneration, and we discuss the possible effects that changes in phospholipid metabolism could play in the neuropathophysiology of Sandhoff disease.

Animals↗

PubMed2004

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase.

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.

Amino Acid Substitution↗

PubMed2003

Purification and characterization of recombinant, human acid ceramidase. Catalytic reactions and interactions with acid sphingomyelinase.

Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(alpha) and 40 (beta)-kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct beta-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the approximately 2-4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.

Adenoviridae↗

PubMed2003

Biosynthesis and degradation of mammalian glycosphingolipids.

Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.

Animals↗

PubMed2003

The reverse activity of human acid ceramidase.

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.

Acid Ceramidase↗

PubMed2003

The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease.

Human lysosomal beta-hexosaminidases are dimeric enzymes composed of alpha and beta-chains, encoded by the genes HEXA and HEXB. They occur in three isoforms, the homodimeric hexosaminidases B (betabeta) and S (alphaalpha), and the heterodimeric hexosaminidase A (alphabeta), where dimerization is required for catalytic activity. Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay-Sachs disease and Sandhoff disease, respectively. Here, we present the crystal structure of a complex of human beta-hexosaminidase B with a transition state analogue inhibitor at 2.3A resolution (pdb 1o7a). On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by beta-hexosaminidase B is proposed. In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation. The structure reported here is a valid template also for the dimeric structures of beta-hexosaminidase A and S.

Acetylglucosamine↗

PubMed2003

Detection and identification of Vav1 protein in primary cultured murine cerebellar neurons and in neuroblastoma cells (SH-SY5Y and Neuro-2a).

Vav1 was detected in neuronal cells during a screening for 1-methylthiodihydroceramide (1-MSDH-Cer) binding proteins. 1-MSDH-Cer is a metabolically stable analogue of dihydroceramide that was reported to strongly interfere with the formation of ceramide and hence the biosynthesis of all sphingolipids in neuronal cells. To identify target proteins that function as putative mediators of this molecule, a 1-MSDH-Cer affinity chromatography was utilised. When the cytosolic fraction of human neuroblastoma SH-SY5Y cells was subjected to 1-MSDH-Cer affinity chromatography, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the eluted protein fraction revealed an about 2-fold enrichment of the 98 kD protein band. Tryptic digestion of the excised band in combination with MALDI mass spectrometry strongly suggested that this band contained Vav1 protein. This was surprising since Vav1 in contrast to the other two isoforms Vav2 and Vav3 is believed to be exclusively expressed in hematopoietic cells and has not been detected in neuronal cells until now. The expression of Vav1 was confirmed in human SH-SY5Y neuroblastoma cells and additionally in murine Neuro-2A neuroblastoma cells as well as in primary cultured murine cerebellar neurons by Western blot analysis and reverse transcription polymerase chain reaction.

Animals↗