Search

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Deficient recovery from acute kidney injury (AKI) has immediate and long-term health, clinical and economic consequences. Pre-emptive recovery estimation may improve nephrology referral, optimize decision making, enrollment in trials, and provide key information for subsequent clinical handling and follow-up. For this purpose, new biomarkers are needed that predict outcome during the AKI episode. We hypothesized that damage pattern-specific biomarkers are expected to more closely associate to outcome within distinct subpopulations (i.e. those affected by specific pathological processes determining a specific outcome), as biomarker pleiotropy (i.e. associated to phenomena unrelated to AKI) introduced by unselected, heterogeneous populations may blur statistics. A panel of urinary biomarkers was measured in patients with AKI and their capacity to associate to normal or abnormal recovery was studied in the whole cohort or after sub-classification by AKI etiology, namely pre-renal and intrinsic AKI. A combination of urinary GM2AP and TCP1-eta best associates with recovery from AKI, specifically within the sub-population of renal AKI patients. This two-step strategy generates a multidimensional space in which patients with specific characteristics (i.e. renal AKI patients with good or bad prognosis) can be identified based on a collection of biomarkers working serially, applying pathophysiology-driven criteria to estimate AKI recovery, to facilitate pre-emptive and personalized handling.

The catabolism of ganglioside GM2 is dependent on three gene products. Mutations in any of these genes result in a different type of GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease, and the B1 and AB variants of GM2 gangliosidosis), with GM2 as the major lysosomal storage compound. GM2 is also a secondary storage compound in lysosomal storage diseases such as Niemann-Pick disease types A–C, with primary storage of SM in type A and cholesterol in types B and C, respectively. The reconstitution of GM2 catabolism at liposomal surfaces carrying GM2 revealed that incorporating lipids into the GM2-carrying membrane such as cholesterol, SM, sphingosine, and sphinganine inhibits GM2 hydrolysis by α-hexosaminidase A assisted by GM2 activator protein, while anionic lipids, ceramide, fatty acids, lysophosphatidylcholine, and diacylglycerol stimulate GM2 catabolism. In contrast, the hydrolysis of the synthetic, water-soluble substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-α-d-glucopyranoside was neither significantly affected by membrane lipids such as ceramide or SM nor stimulated by anionic lipids such as bis(monoacylglycero)phosphate added as liposomes, detergent micelles, or lipid aggregates. Moreover, hydrolysis-inhibiting lipids also had an inhibiting effect on the solubilization and mobilization of membrane-bound lipids by the GM2 activator protein, while the stimulating lipids enhanced lipid mobilization.—

In this work, we report, for the first time, the construction of a label-free electrochemical immunosensor for highly sensitive detection of a new lung cancer biomarker, GM2 activator protein (GM2AP). A polyethyleneimine-coated gold nanoparticle (PEI-AuNP) and phosphomolybdic acid (PMA) modified electrode is developed as a novel redox platform for GM2AP detection. A PEI-AuNP film-modified screen-printed carbon electrode, as a signal amplifier support, was successfully fabricated for the adsorption of PMA redox molecules and is used for signal amplification. Under the optimized conditions, GM2AP detection is based on a decrease in the current response of PMA redox probes proportionally relative to an amount of the immunocomplex. Our sensor exhibits two linear ranges of 0.005-25 and 25-400 ng mL-1 with a limit of detection (LOD) of 0.51 pg mL-1. The immunosensor is successfully applied for the determination of GM2AP in both human urine and serum samples. The proposed sensor offers the advantages of simple fabrication, low cost, rapid analysis, satisfactory stability, high selectivity and sensitivity, and good reproducibility. The LOD of the biosensor is approximately 2863 and 1804 fold lower than the clinically relevant levels in human urine and serum, respectively. Our strategy can be used as an alternative non-invasive clinical analysis method for lung cancer screening.

GM2 gangliosidosis is a rare form of inborn errors of metabolism including Tay-Sachs disease, Sandhoff disease, and GM2 activator deficiency. GM2 activator protein deficiency is an ultra-rare form of GM2 gangliosidosis. To date, 16 cases of GM2 activator protein deficiency have been reported in the literature, and among them, 11 cases were the infantile form of the disease. Here we report the first two patients from Turkey with the infantile form of the disease with a novel likely pathogenic variant.A boy of eight months old presented to the metabolic department with very mild neurological deterioration, although he had achieved early developmental milestones at the appropriate time. The parents also had a daughter who had lost skills progressively before one year of age. The boy was evaluated and bilateral cherry-red spots were found with no abnormality in either metabolic screening including β-hexosaminidase or cranial magnetic resonance imaging. A novel homozygous likely pathogenic variant inThis case report emphasizes that in the event of normal β-hexosaminidase activity, GM2 activator protein deficiency could be underdiagnosed, and further molecular analysis should be performed. To the best of our knowledge, this boy is one of the youngest patient diagnosed with very mild symptoms. With this novel pathogenic variant, these patients have expanded the mutation spectrum of GM2 activator protein deficiency.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g. intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

Renal dysfunction, a major complication of type 2 diabetes, can be predicted from estimated glomerular filtration rate (eGFR) and protein markers such as albumin concentration. Urinary protein biomarkers may be used to monitor or predict patient status. Urine samples were selected from patients enrolled in the retrospective diabetic kidney disease (DKD) study, including 35 with good and 19 with poor prognosis. After removal of albumin and immunoglobulin, the remaining proteins were reduced, alkylated, digested, and analyzed qualitatively and quantitatively with a nano LC-MS platform. Each protein was identified, and its concentration normalized to that of creatinine. A prognostic model of DKD was formulated based on the adjusted quantities of each protein in the two groups. Of 1296 proteins identified in the 54 urine samples, 66 were differentially abundant in the two groups (area under the curve (AUC): p-value &lt, 0.05), but none showed significantly better performance than albumin. To improve the predictive power by multivariate analysis, five proteins (ACP2, CTSA, GM2A, MUC1, and SPARCL1) were selected as significant by an AUC-based random forest method. The application of two classifiers&mdash, support vector machine and random forest&mdash, showed that the multivariate model performed better than univariate analysis of mucin-1 (AUC: 0.935 vs. 0.791) and albumin (AUC: 1.0 vs. 0.722). The urinary proteome can reflect kidney function directly and can predict the prognosis of patients with chronic kidney dysfunction. Classification based on five urinary proteins may better predict the prognosis of DKD patients than urinary albumin concentration or eGFR.

IntroductionTobacco causes kidney damage that can progress to chronic kidney disease. However, the diagnostic parameters used in clinics are not effective in identifying smokers at risk. Our first objective is to more effectively detect subclinical renal damage in smokers. In addition, we hypothesise that tobacco consumption can predispose smokers to renal damage on exposure to other potentially nephrotoxic events (drugs, diagnostic procedures and so on). We will test this hypothesis in our second objective by investigating whether certain predisposition markers (GM2 ganglioside activator protein (GM2AP), transferrin and t-gelsolin) are able to detect smokers who are predisposed to kidney damage. Finally, in our third objective, we will study whether smoking cessation reduces subclinical and/or predisposition to renal damage.Methods and analysisFor our first objective, a prospective cross-sectional study will be carried out with patients from a primary healthcare centre. The influence of tobacco on renal damage, in patients both with and without additional risk factors, will be studied using a panel of early biomarkers (albuminuria, N-acetyl-beta-D-glucosaminidase, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). For our second objective, a prospective longitudinal study will be carried out with patients recruited for our first objective. We will study whether certain predisposition biomarkers (GM2AP, transferrin and t-gelsolin) are able to detect smokers predisposed to renal damage. For our third objective, a prospective longitudinal study will be carried out with patients from a smoking cessation unit. We will study the evolution of the markers described above following smoking cessation.Ethics and disseminationThe study has been approved by the Clinical Research Ethics Committee of the Healthcare Area of Salamanca. All study participants will sign an informed consent form in compliance with the Declaration of Helsinki and the WHO standards for observational studies. Results will be presented at conferences and submitted to peer-reviewed journals.Trial registration numberNCT03850756.

The GM2-activator protein (GM2-AP), revealed by Li et al. in 1973 in human liver, was initially identified as a protein cofactor that stimulated β-hexosaminidase A to hydrolyze N-acetylgalactosamine from GM2 ganglioside. This cofactor was found to be missing in human variant AB Tay-Sachs disease. Over the years, the GM2-AP has also been shown to be involved in kidney vesicular transport, lipid presentation by CD1 molecule to T-cells, and interaction of human sperm with zona pellucida. Since the expression of the GM2-AP via mRNA detection in mouse tissues was found to be the highest in testis, we became interested in the localization of the GM2-AP at cellular level in mouse testis during spermatogenesis. Using immunohistochemical analysis and electron microscopy, we found that the GM2-AP was predominantly localized in the basal cytoplasm and the attenuated processes of Sertoli cells. The stained structure appeared to be lysosomes. The most interesting finding was the association of the GM2-AP with the acrosomal apparatus in early spermatids. A modest to intense staining was observed in some acrosomal granules and acrosomal caps. The GM2-AP seemed to disappear from acrosomal caps in the later stage of spermatids, in which the nucleus became elongated and condensed. These results suggest that the GM2-AP may be involved in the normal functions of Sertoli cells and play important roles during the development of acrosomal caps in the early spermatids.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as “River blindness”, a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O. volvulus ESPs to address the biology of this parasite. We showed that (1) Ov28CRP is a putative ganglioside GM2 Activator Protein (GM2AP) conserved in nematode; (2) OvGM2AP gene is transcriptionally activated in all investigated stages of the parasitic life cycle, including larval and adult stages; (3) The full-length OvGM2AP was detected in in-vitro O. volvulus ESPs of adult and larval stages; (4) the mass expressed and purified recombinant OvGM2AP purified from insect cell culture medium was found to be glycosylated at asparagine 173 and lacked N-terminal signal peptide sequence; (5) the recombinant OvGM2AP discriminated serum samples of infected and uninfected individuals; (6) OvGM2AP competitively inhibits MUG degradation by recombinant β-hexosaminidase A but not MUGS, and could not hydrolyze the GM2 to GM3; (7) humoral immune responses to the recombinant OvGM2AP revealed a negative correlation with ivermectin treatment. Altogether, our findings suggest for the first time that OvGM2AP is an antigenic molecule whose biochemical and immunological features are important to gain more insight into our understanding of host-parasite relationship, as well as its function in parasite development at large., SCOPUS: ar.j, info:eu-repo/semantics/published

GRP94 is an ATP-dependent chaperone able to regulate pro-oncogenic signaling pathways. Previous studies have shown a critical role of GRP94 in brain metastasis (BrM) pathogenesis and progression. In this work, an untargeted lipidomic analysis revealed that some lipid species were altered in GRP94-deficient cells, specially GM2 and GM3 gangliosides. The catalytic pathway of GM2 is affected by the low enzymatic activity of β-Hexosaminidase (HexA), responsible for the hydrolysis of GM2 to GM3. Moreover, a deficiency of the GM2-activator protein (GM2-AP), the cofactor of HexA, is observed without alteration of gene expression, indicating a post-transcriptional alteration of GM2-AP in the GRP94-ablated cells. One plausible explanation of these observations is that GM2-AP is a client of GRP94, resulting in defective GM2 catabolic processing and lysosomal accumulation of GM2 in GRP94-ablated cells. Overall, given the role of gangliosides in cell surface dynamics and signaling, their imbalance might be linked to modifications of cell behaviour acquired in BrM progression. This work indicates that GM2-AP could be an important factor in ganglioside balance maintenance. These findings highlight the relevance of GM3 and GM2 gangliosides in BrM and reveal GM2-AP as a promising diagnosis and therapeutic target in BrM research. © 2019, The Author(s)., This work was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 35 no. 320737, Generalitat de Catalunya (Suport a les activitats de Grups de Recerca, 2017 SGR 753), and Spanish Ministry of Health and Consumer Affairs FIS-PI14/00336 and FIS-PI18/00916 grants from the I + D + I National Plan with the financial support from ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). The authors also thank Joan Meléndez for illustration and infographic support.

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington’s disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington’s Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington’s disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.

Alcohol dependence (AD) and nicotine dependence (ND) co-occur frequently (AD+ND). We integrated SNP-based, gene-based, and protein-protein interaction network analyses to identify shared risk genes or gene subnetworks for AD+ND in African Americans (AAs, N = 2,094) and European Americans (EAs, N = 1,207). The DSM-IV criterion counts for AD and ND were modeled as two dependent variables in a multivariate linear mixed model, and analyzed separately for the two populations. The most significant SNP was rs6579845 in EAs (p < 1.29 × 10-8 ) in GM2A, which encodes GM2 ganglioside activator, and is a cis-expression quantitative locus that affects GM2A expression in blood and brain tissues. However, this SNP was not replicated in our another small sample (N = 678). We identified a subnetwork of 24 genes that contributed to the AD+ND criterion counts. In the gene-set analysis for the subnetwork in an independent sample, the Study of Addiction: Genetics and Environment project (predominately EAs), these 24 genes as a set differed in AD+ND versus control subjects in EAs (p = .041). Functional enrichment analysis for this subnetwork revealed that the gene enrichment involved primarily nerve growth factor pathways, and cocaine and amphetamine addiction. In conclusion, we identified a genome-wide significant variant at GM2A and a gene subnetwork underlying the genetic trait of shared AD+ND. These results increase our understanding of the shared (pleiotropic) genetic risk that underlies AD+ND.

The aim of the present study was to investigate the association of neonatal necrotizing enterocolitis (NEC) with myeloid differentiation-(MD-2) and GM2 activator protein (GM2A) genetic polymorphisms. Gene resequencing of the MD-2 and GM2A gene exons was performed on 42 neonates, diagnosed with NEC (NEC group), as well as in the rs11465996 locus, located in the MD-2 gene promoter region. The aim was to detect the genetic polymorphisms present in the neonates with NEC and compare the functional polymorphic loci with 83 neonates without NEC (control group), who had been born during the same period. A polymorphic locus with abnormal frequency was detected in the exon region of the MD-2 gene. In the NEC group, the frequency of genotypes carrying the low frequency allele (G) in the rs11465996 locus (MD-2 promoter region) was significantly higher compared with the control group (χ(2)=4.388, P=0.036). Furthermore, the frequencies of genotypes carrying the low frequency A and C alleles in the rs1048719 (GM2A gene exon 1) and rs2075783 loci (GM2A intron), respectively, were significantly higher in the NEC group compared with the control group (χ(2)=4.316, P=0.038; and χ(2)=13.717, P=0.000, respectively). In addition, the rs11465996 polymorphism in the MD-2 gene promoter region was found to be associated with the severity of NEC. Furthermore, the rs2075783 polymorphism in the GM2A gene exon 1 and the rs1048719 polymorphism in the intron region of this gene, were associated with the occurrence of NEC. The present study demonstrated that gene polymorphisms of MD-2 and GM2A were associated with the occurrence or severity of NEC; however, further in-depth exploration is required to clarify the associations between genetic predispositions to polymorphisms, and NEC.

IntroductionEffective biomarkers for early diagnosis of lung cancer are needed. A recent study demonstrated that urinary GM2-activator protein (GM2AP) level was increased in lung cancer patients. This study aims to validate the potential application of GM2AP as a biomarker for diagnosis of lung cancer.MethodsSerum and urine samples were obtained from 189 participants (133 patients for treatment naive lung cancer, 26 healthy volunteers for urine, and 30 healthy volunteers for serum). GM2AP level was detected by Western blotting and quantified using enzyme-linked immunosorbent assay (ELISA). The GM2AP expression in tumors and nontumor parts of lung tissues from 143 nonsmall cell lung cancers was detected by immunohistochemical stains.ResultsThere was an 8.11 ± 1.36 folds increase in urine and a 5.41 ± 0.73 folds increase in serum level of GM2AP in lung cancer patients compared with healthy volunteers (p < 0.0001), achieving a 0.89 AUC value in urine and 0.90 AUC value in serum for the receiver-operating characteristic curves. Both serum and urine levels of GM2AP correlated significantly with pathology stages (urine, p = 0.009; serum, p < 0.0001). Using immunohistochemical, positive expression of GM2AP was found at 83.9% of nonsmall cell lung cancers patients and none in normal tissue. The GM2AP expression was significantly correlated with pathology stage (p = 0.0001). Patients with higher GM2AP expression had shorter overall survival (p = 0.045) and disease-free survival (p = 0.049) than lower GM2AP expression. Moreover, the multivariate analysis suggested GM2AP as an independent predictors of disease-free survival and overall survival.ConclusionsOur study demonstrates that GM2AP might serve as potential diagnostic and prognostic biomarkers in patients with lung cancer.

β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 A revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of β-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering. DATABASE Structural data are available in the PDB database under the accession number 5OAR. ENZYME β-N-acetylhexosaminidase (EC 3.2.1.52).

GM2 gangliosidosis, AB variant, is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein. We report on two patients with typical clinical features suggestive of GM2 gangliosidosis, but normal results for hexosaminidase A and hexosaminidase B as well as their corresponding genes. Genetic analysis of the gene encoding the activator protein, the GM2A gene, elucidated the cause of the disease, adding a novel mutation to the spectrum of GM2 AB variant. This report points out that in typical clinical constellations with normal enzyme results, genetic diagnostic for activator protein defects should be performed.

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

We describe a novel peptide ligation/desulfurization strategy using a β-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly.

Sandhoff disease is a GM2 gangliosidosis caused by mutations in HEXB encoding the β-subunit of β-hexosaminidase A. β-Hexosaminidase A exists as a heterodimer consisting of α- and β-subunits, and requires a GM2 activator protein to hydrolyze GM2. To investigate the molecular pathology in an adult Sandhoff disease patient with an early disease onset, we performed mutation detection, western blot analysis and molecular simulation analysis. The patient had compound heterozygous mutations p.Arg505Gln and p.Ser341ValfsX30. Western blot analysis showed that the amount of mature form of the α- and β-subunits was markedly decreased in the patient. We then performed docking simulation analysis of the α- and β-subunits with p.Arg505Gln, the GM2AP/GM2 complex and β-hexosaminidase A, and GM2 and β-hexosaminidase A. Simulation analysis showed that p.Arg505Gln impaired each step of molecular conformation of the α- and β-subunits heterodimer, the activator protein and GM2. The results indicated that p.Ser341ValfsX30 reduced the amount of β-subunit, and that p.Arg505Gln hampered the maturation of α- and β-subunits, and hindered the catalytic ability of β-hexosaminidase A. In conclusion, various methods including simulation analysis were useful to understand the molecular pathology in Sandhoff disease.

Using label-free quantative proteomics, we have identified 2 potential protein biomarkers that indicate presymptomatic kidney disease in the urine of pediatric patients with type-I diabetes and Fabry disease (n = 20). Prosaposin and GM2 activator protein (GM2AP) were observed to be elevated in the urine of these patient groups compared to age- and sex-matched controls. These findings were validated by development of a rapid MRM-based tandem mass spectrometry test. Prosaposin was observed to be both significantly elevated in the urine of patients with Fabry disease compared to controls (p = 0.02) and reduced after 12 months enzyme replacement therapy (ERT, p = 0.01). Similarly, GM2AP concentrations were observed to be significantly higher compared to controls in the diabetic group (p = 0.049) and the pretreatment Fabry group (p = 0.003). In addition, this observed to be reduced significantly in the Fabry group following 12 months of ERT (p = 0.01). The process of detection of the biomarkers, development into a test and implications for monitoring patients and treatment are discussed.

A synthetic protocol for the preparation of 162-residue S-monoglycosylated GM2-activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one-pot/N-to-C-directed sequential ligation strategy using readily accessible middle N-sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1-31)] with the N-terminal cysteinyl prolyl thioester [GM2AP (32-67)] proceeded smoothly to yield the 67-residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as "tailored synthesis" for the construction of a GM2AP library.

Adipose tissue has profound effects on whole-body insulin sensitivity. However, the underlying biological processes are quite complex and likely multifactorial. For instance, the adipose transcriptome is posttranscriptionally modulated by microRNAs, but the relationship between microRNAs and insulin sensitivity in humans remains to be determined. To this end, we utilized an integrative mRNA-microRNA microarray approach to identify putative molecular interactions that regulate the transcriptome in subcutaneous adipose tissue of insulin-sensitive (IS) and insulin-resistant (IR) individuals. Using the NanoString nCounter Human v1 microRNA Expression Assay, we show that 17 microRNAs are differentially expressed in IR vs. IS. Of these, 16 microRNAs (94%) are downregulated in IR vs. IS, including miR-26b, miR-30b, and miR-145. Using Agilent Human Whole Genome arrays, we identified genes that were predicted targets of miR-26b, miR-30b, and miR-145 and were upregulated in IR subjects. This analysis produced ADAM22, MYO5A, LOX, and GM2A as predicted gene targets of these microRNAs. We then validated that miR-145 and miR-30b regulate these mRNAs in differentiated human adipose stem cells. We suggest that use of bioinformatic integration of mRNA and microRNA arrays yields verifiable mRNA-microRNA pairs that are associated with insulin resistance and can be validated in vitro.

Cancer cell secretomes are considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF-7, MDA-MB-231, and SK-BR-3) were profiled with liquid chromatography-tandem mass spectrometry analysis. A total of 1410 proteins were identified with less than 1% false discovery rate, of which approximately 55% (796 proteins) were predicted to be secreted from cells. To find BC-specific proteins among the secreted proteins, data of immunohistochemical staining compiled in the Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion, and sufficient number of tandem mass spectra, 12 biomarker candidate proteins including ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot analysis and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially those who were estrogen receptor-negative. Additionally, siRNA-mediated knockdown of GM2A in BC cells decreased migration in vitro, whereas the overexpression of GM2A led to an increase in cell migration. Although GM2A as a diagnostic and prognostic marker in BC should be carefully verified further, this study has established the potential role of GM2A in BC progression.

Recently, we identified ganglioside GM2 activator protein (GM2AP) as a novel adipokine, and revealed that treatment of cultured cells with GM2AP impairs insulin signal transduction. The aim of this study was to examine the impact of GM2AP on glucose metabolism in vivo. Injection of recombinant GM2AP in mice significantly lowered blood glucose levels in glucose tolerance tests. Administration of GM2AP to mice for 10days increased serum insulin levels, whereas the contents of glucose, leptin and FFA were significantly decreased. Stimulation of calcium influx and insulin secretion by GM2AP was observed in hamster insulinoma HIT-T15 cells. Blockage of GM2AP function by specific antibodies inhibited GM2AP-induced insulin secretion. These results provide novel insights into the physiological functions of GM2AP in obesity.

In an attempt to discover novel adipokines, we performed proteomics analyses using culture medium from differentiated 3T3-L1 adipocytes, and first identified GM2AP. The levels of GM2AP mRNA and protein were augmented by adipogenesis in cultured adipocytes and expression in adipose tissue and serum of obese mice or human subjects was found to be significantly higher than in lean counterparts. Exposure of 3T3-L1 adipocytes to GM2AP protein accelerated dissociation of insulin receptor-beta (IRβ) from caveolin-1, and interrupted insulin signal transduction. Abrogation of GM2AP function by specific antibodies augmented glucose uptake. Furthermore, treatment of rat pheochromocytoma PC12 NS1 cells with GM2AP impaired NGF signal transduction. Taken together, these results provide novel insights into the physiological functions of GM2AP in obesity.

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann–Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Platelet activating factor (PAF), an endogenous bioactive phospholipid, has been documented as a pivotal mediator in the inflammatory cascade underlying the pathogenesis of many diseases including necrotizing enterocolitis. Much effort has been directed towards finding an effective in vivo inhibitor of PAF signaling. Here, we report that a small, highly stable, lysosomal lipid transport protein, the GM2 activator protein (GM2AP) is able to inhibit the inflammatory processes otherwise initiated by PAF in a rat model of necrotizing enterocolitis. Based on behavioral observations, gross anatomical observations at necropsy, histopathology and immunocytochemistry, the administration of recombinant GM2AP inhibits the devastating gastrointestinal necrosis resulting from the injection of rats with LPS and PAF. Recombinant GM2AP treatment not only markedly decrease tissue destruction, but also helped to maintain tight junction integrity at the gastrointestinal level as judged by contiguous Zonula Occludens-1 staining of the epithelial layer lining the crypts.

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Forster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.

G M2 -gangliosidoses are neurodegenerative storage diseases, associated with intraneuronal G M2 -ganglioside accumulation. Intralysosomal G M2 -ganglioside hydrolysis requires the concerted action of β-hexosamidase A (HexA) and its cofactor G M2 activator protein. Thus, disease can occur as a consequence of mutations in either the gene which encodes subunits of HexA (α and β) or its cofactor, each of which is mapped to independent loci. There is significant overlap in clinical presentation among these three clinical subtypes, encompassing Tay–Sachs disease (α-mutations), Sandhoff disease (β-mutations), and the AB variant (cofactor defect), all of which are inherited in an autosomal recessive fashion. The majority of affected individuals display a rapid degenerative course in childhood, and there are late-onset forms associated with life extending into decades. Ongoing studies of underlying pathogenesis implicate neuroinflammatory processes. There are investigations of various therapeutic approaches, including substrate reduction therapy, the use of pharmacologic chaperones and gene therapy. However, no specific treatment has been introduced that has altered what is ultimately a poor prognosis. Prevention has been achieved through carrier testing in populations at risk and prenatal diagnosis.

We have identified a GM2-activator protein (GM2AP) with highly unusual properties secreted by the nematode parasite Trichinella spiralis . Expression in Pichia pastoris resulted in a hyperglycosylated protein of 28 kDa, but the 18 kDa native protein was not glycosylated. The parasite GM2AP does not facilitate degradation of GM2 ganglioside by N -acetyl-β-hexosaminidase A, although it does inhibit phospholipase D activity. Lack of the former activity might be explained by the absence of a domain implicated in binding to hexosaminidase. In addition, and contrary to data on the human GM2AP, the nematode homologue does not inhibit platelet activating factor-induced calcium mobilisation in neutrophils, but actually enhances mediator-induced chemotaxis.

The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-d-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme. Germany.

We previously found that oxidized low-density lipoprotein (LDL) elevated the expression of mRNA of GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (GM2) ganglioside activator protein, in human monocyte-derived macrophages. Recently, GM2 activator protein has become known as a general glycosphingolipid transporter as well as a specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. Accumulation of glycosphingolipids has been observed in the serum or aorta of atherosclerotic model animals and humans. The proliferation of aortic smooth muscle cells, elevation of LDL uptake by macrophages, interfering LDL clearance by the liver, and enhancement of platelet adhesion to collagen have been proposed as the underlying mechanisms of glycosphingolipid-mediated atherogenesis. The GM2 activator protein can bind, solubilize and transport a broad spectrum of lipid molecules, indicating that GM2 activator protein may function as a general intra- and inter-cellular lipid transport protein. Collectively, elevated levels of GM2 activator protein in the aorta may be another feature of human atherosclerosis.

The GM2 gangliosidoses are a group of autosomal recessive lysosomal storage disorders caused by defective β-hexosaminidase. There are three clinical conditions in this group: Tay-Sachs disease (TSD), Sandhoff disease (SD), and hexosaminidase activator deficiency. The three conditions are clinically indistinguishable. TSD and SD have been identified with infantile, juvenile, and adult onset forms. The activator deficiency is only known to present with infantile onset. Diagnosis of TSD and SD is based on decreased hexosaminidase activity and a change in the percentage of activity between isoforms. There are no biochemical tests currently available for activator deficiency. This unit provides a detailed procedure for identifying TSD and SD in affected individuals and carriers from leukocyte samples, the most robust sample type available.

The G(M2) activator protein is required for successful degradation of G(M2) ganglioside by the A isozyme of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52). Deficiency of the G(M2) activator protein leads to a relentlessly progressive accumulation of G(M2) ganglioside in neuronal lysosomes and subsequent fatal deterioration of central nervous system function. G(M2) activator deficiency has been described in humans, dogs and mice. This manuscript reports the discovery and characterization of a feline model of G(M2) activator deficiency that exhibits many disease traits typical of the disorder in other species. Cats deficient in the G(M2) activator protein develop clinical signs at approximately 14 months of age, including motor incoordination and exaggerated startle response to sharp sounds. Affected cats exhibit central nervous system abnormalities such as swollen neurons, membranous cytoplasmic bodies, increased sialic acid content and elevated levels of G(M2) ganglioside. As is typical of G(M2) activator deficiency, hexosaminidase A activity in tissue homogenates appears normal when assayed with a commonly used synthetic substrate. When the G(M2) activator cDNA was sequenced from normal and affected cats, a deletion of 4 base pairs was identified as the causative mutation, resulting in alteration of 21 amino acids at the C terminus of the G(M2) activator protein.

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.

The formation of neurotoxic beta-amyloid fibrils in Alzheimer's disease (AD) is suggested to involve membrane rafts and to be promoted, in vitro, by enriched concentrations of gangliosides, particularly GM1, and the cholesterol therein. In our study, the presence of rafts and their content of the major membrane lipids and gangliosides in the temporal cortex, reflecting late stages of AD pathology, and the frontal cortex, presenting earlier stages, has been investigated. Whole tissue and isolated detergent-resistant membrane fractions (DRMs) were analysed from 10 AD and 10 age-matched control autopsy brains. DRMs from the frontal cortex of AD brains contained a significantly higher concentration (micromol/micromol glycerophospholipids), of ganglioside GM1 (22.3 +/- 4.6 compared to 10.3 +/- 6.4, p

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2 A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.

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.

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. D-53121 Bonn, Germany.

The GM2-activator protein (GM2-AP) is a small lysosomal lipid transfer protein essential for the hydrolytic conversion of ganglioside GM2 to GM3 by beta-hexosaminidase A. The crystal structure of human apo-GM2-AP is known to consist of a novel beta-cup fold with a spacious hydrophobic interior. Here, we present two new structures of GM2-AP with bound lipids, showing two different lipid-binding modes within the apolar pocket. The 1.9A structure with GM2 bound shows the position of the ceramide tail and significant conformational differences among the three molecular copies in the asymmetric unit. The tetrasaccharide head group is not visible and is presumed to be disordered. However, its general position could be established through modeling. The structure of a low-pH crystal, determined at 2.5A resolution, has a significantly enlarged hydrophobic channel that merges with the apolar pocket. Electron density inside the pocket and channel suggests the presence of a trapped phospholipid molecule. Structure alignments among the four crystallographically unique monomers provide information on the potential role for lipid binding of flexible chain segments at the rim of the cavity opening. Two discrete orientations of the S130-T133 loop define an open and a closed configuration of the hydrophobic channel that merges with the apolar pocket. We propose: (i) that the low-pH structure represents an active membrane-binding conformation; (ii) that the mobile S130-T133 loop serves as a gate for passage of ligand into the apolar pocket; and (iii) that this loop and the adjacent apolar V59-W63 loop form a surface patch with two exposed tryptophan residues that could interface with lipid bilayers.

GM2 activator protein (GM2AP) is a specific protein cofactor that stimulates the enzymatic hydrolysis of the GalNAc from GM2, a sialic acid containing glycosphingolipid, both in vitro and in lysosomes. While phospholipids together with glycosphingolipids are important membrane constituents, little is known about the possible effect of GM2AP on the hydrolysis of phospholipids. Several recent reports suggest that GM2AP might have functions other than stimulating the conversion of GM2 into GM3 by beta-hexosaminidase A, such as inhibiting the activity of platelet activating factor and enhancing the degradation of phosphatidylcholine by phospholipase D (PLD). We therefore examined the effect of GM2AP on the in vitro hydrolyses of a number of phospholipids and sphingomyelin by microbial (Streptomyces chromofuscus) and plant (cabbage) PLD. GM2AP, at the concentration as low as 1.08 microM (1 microg/50 microl) was found to inhibit about 70% of the hydrolyses of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol by PLD, whereas the same concentration of GM2AP only inhibited about 20-25% of the hydrolysis of sphingomyelin by sphingomyelinase and had no effect on the hydrolysis of sphingosylphosphorylcholine by PLD. Thus, GM2AP exerts strong and broad inhibitory effects on the hydrolysis of phospholipids carried out by plant and microbial PLDs. High ammonium sulfate concentration (1.6 M or 21.1%) masks this inhibitory effect, possibly due to the alteration of the ionic property of GM2AP.

GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by β-hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized α-helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2.

Human lysosomal beta-hexosaminidases remove terminal beta-glycosidically bound N-acetylhexosamine residues from a number of glycoconjugates. Three different isozymes composed of two noncovalently linked subunits alpha and beta exist: Hex A (alphabeta), Hex B (betabeta), and Hex S (alphaalpha). While the role of Hex A and B for the degradation of several anionic and neutral glycoconjugates has been well established, the physiological significance of labile Hex S has remained unclear. However, the striking accumulation of anionic oligosaccharides in double knockout mice totally deficient in hexosaminidase activity but not in mice expressing Hex S (Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C.J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L., (1996) Nat. Genet. 14, 348-352) prompted us to reinvestigate the substrate specificity of Hex S. To identify physiological substrates of Hex S, anionic and neutral oligosaccharides excreted in the urine of the double knockout mice were isolated and analyzed. Using ESI-MS/MS and glycosidase digestion the anionic glycans were identified as products of incomplete dermatan sulfate degradation whereas the neutral storage oligosaccharides were found to be fragments of N-glycan degradation. In vitro, recombinant Hex S was highly active on water-soluble and amphiphilic glycoconjugates including artificial substrates, sulfated GAG fragments, and the sulfated glycosphingolipid SM2. Hydrolysis of membrane-bound SM2 by the recombinant Hex S was synergistically stimulated by the GM2 activator protein and the lysosomal anionic phospholipid bis(monoacylglycero)phosphate.

Artificial fusion of human monocyte with Cloudman S91 mouse melanoma cells resulted in hybrids that showed increased motility in vitro, enhanced metastatic potential in vivo, and also tended to be super melanotic (Rachkovsky et al., Clin. Exp. Metastasis 16 (1998) 299). However, no gene derived from monocytes has been shown to be expressed in these hybrids until now. Similar observations have also been noted in hybrids originating from mouse macrophage and mouse melanoma cells. Having the advantage of species differences in mouse×human hybrids, we are able, this time, to show by RT-PCR that some genes specific to the human genome are expressed in these hybrids, indicating that not only is the genomic DNA from parental monocytes integrated in the hybrids but also some genes are being expressed. This observation may lead us to find contributory genes from monocyte and/or macrophage that are responsible for modulating the genotypes and hence the phenotypes in the hybrids.