Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles.

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.

The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma.

RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.

Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants.

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.

Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal.

Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers. In 23 Nepalese pregnant women who delivered at term, two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2-6.8) exhibited nominally significant (p < 0.05) differences in abundance across trimesters. Additional filtering using a Bonferroni correction reduced the number of significant (p < 0.00019) spots to 61. Mass spectrometric analysis detected 38 proteins associated with gestational age, cytoskeletal remodeling, blood pressure regulation, lipid and nutrient transport, and inflammation. One new protein, pregnancy-specific ß-glycoprotein 4 was detected. A follow-up isotope tagging for relative and absolute quantitation (iTRAQ) experiment of six mothers from the DIGE study revealed 111 proteins, of which 11 exhibited significant (p < 0.05) differences between trimesters. Four of these proteins: gelsolin, complement C1r subcomponent, α-1-acid glycoprotein, and α-1B-glycoprotein also changed in the DIGE analysis. Although not previously associated with normal pregnancy, gelsolin decreased in abundance by the third trimester (p < 0.01) in DIGE, iTRAQ and Western analyses. Changes in abundance of proteins in serum that are associated with syncytiotrophoblasts (gelsolin, pregnancy-specific ß-1 glycoprotein 1 and ß-2-glycoprotein I) probably reflect dynamics of a placental proteome shed into maternal circulation during pregnancy. Measurement of changes in the maternal serum proteome, when linked with birth outcomes, may yield biomarkers for tracking reproductive health in resource poor settings in future studies.

A mannose-specific recognition mediates the defasciculation of axons in the leech CNS.

We are studying a mannose-specific recognition mediating the projection of axons in the synaptic neuropil of the embryonic leech CNS. A functional class of neurons, the sensory afferents, can be distinguished by a mannose-containing epitope that is asparagine-linked to a 130 kDa surface protein and is reactive with the monoclonal antibody Lan3-2. Sensory afferents project as a tightly fasciculated bundle through peripheral nerves but, upon arriving in the CNS, defasciculate into the synaptic neuropil. This defasciculation allows the previously bundled sensory afferents to form an arborization in the synaptic neuropil. Three lines of experimental evidence indicate that the defasciculation is mediated by the sensory afferent's mannose-containing Lan3-2 epitope. The defasciculation is inhibited (1) by blocking the Lan3-2 epitope with Lan3-2 Fab fragments, (2) by cleaving the asparagine-linked carbohydrate moieties from surface proteins with the glycosidase N-glycanase, and (3) by competing for a putative mannose-binding protein with the neoglycoprotein mannose-BSA [albumin, p-aminophenyl alpha-D-mannopyranoside (26 mol monosaccharide/mol albumin)]. In addition to inhibiting the defasciculation, the three perturbation reagents also elicited the refasciculation of axons that had defasciculated prior to their application. These three different experimental approaches provide strong evidence that carbohydrate recognition regulates the projections of sensory afferents in the leech synaptic neuropil. Carbohydrate interactions therefore can play a major role in regulating the neuronal architecture in the CNS.

Carbohydrate-binding proteins in the leech: I. Isolation and characterization of lactose-binding proteins.

Three lactose-binding proteins with apparent molecular masses of 16, 35, and 63 kDa [leech lectin 16, 35, and 63 (LL,16, LL35, and LL63, respectively)] were isolated from leech membranes. Polyclonal antibodies raised against LL35 cross-reacted with LL16 and LL63, indicating that all three lectins were immunologically related. These leech lectins, however, can be subdivided into two groups based on their tissue distributions and binding affinities for galactose derivatives. LL16 and LL35 are endogenous to the leech's CNS, whereas LL63 is only present in peripheral organs. LL16 and LL35, found in the CNS, bind both the alpha and beta anomers of methylgalactose, whereas the peripheral lectin LL63 binds only the beta form. LL35 and LL63 also differ in their binding affinities for galactosamine and N-acetylgalactosamine. The binding activity of LL35 was calcium independent and active over a wide pH range. Triton X-100 and 2-mercaptoethanol were necessary to recover LL35 binding activity during extraction. These characteristics strongly suggest that LL35 is another member of the calcium-independent galactose/lactose-specific lectins previously described in vertebrates and recently demonstrated in sponges and nematodes. Because a single leech expresses up to 100 micrograms of LL35, this leech lectin is readily amenable to structural and functional analysis.

Carbohydrate-binding proteins in the leech: II. Lactose-binding protein LL35 is located to neuronal and muscle subsets and all epithelial cells.

Leech lectin 35 (LL35) is a calcium-independent galactoside-binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium-independent, galactose-specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity-purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.

Cytosolic O-glycosylation is abundant in nerve terminals.

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modification: beta-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc(1)). O-GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the two enzymes regulating O-GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O-GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron-specific O-GlcNAc modified proteins: collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1) and beta-synuclein. CRMP-2, which is involved in growth cone collapse, is a major O-GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O-GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O-GlcNAc may play a role in neurodegenerative disease.

Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions.

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.

Glial processes, identified through their glial-specific 130 kD surface glycoprotein, are juxtaposed to sites of neurogenesis in the leech germinal plate.

Glial processes, bearing a unique 130 kD surface protein, are located at key sites of morphogenic movement and neuronal differentiation in the leech germinal plate. A midline glial fascicle resides at the primary axis of embryonic symmetry, alongside which teloblasts move as they generate their bandlets of stem cells. The n-bandlets straddle the midline glia and are known to produce most of the central neuroblasts. The midline glia then defasciculates as neuroblasts begin to aggregate into neuromeres. The defasciculated processes expand into these neuromeres, molding the future central neuropile. Neuroblasts will initiate primary axons toward the midline glia. As the neuromeres mature, midline glial process thin out to demarcate the orientation of the future connectives, which are the major longitudinal axon tracts along the midline. Next, segmental but still primordial glia appear in the neuromeres. Initially, they also project longitudinally, then transversely, demarcating the other two major axonal pathways--the central commissures and peripheral roots. Finally, macroglial processes proliferate as massive axon growth invades the central and peripheral nervous system. Thus, glial processes with different developmental histories accompany different aspects of leech neurogenesis. In other systems, glia have been shown to promote the differentiation and the guidance of neurons. It remains to be seen whether the glial-specific 130 kD protein is a receptor mediating these typical glial functions in the leech germinal plate.

The specificity of 130-kDa leech sensory afferent proteins is encoded by their carbohydrate epitopes.

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.

Proton transfer in the mechanism of triosephosphate isomerase.

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate. During the TIM reaction, approximately 2% of the pro-R tritium from C1 of DHAP is conserved and appears at C2 of GAP [Nickbarg, E. B., and Knowles, J. R. (1988) Biochemistry 27, 5939]. In the "classical" mechanism, 98% of the pro-R tritium exchanges with solvent from Glu-165 at the intermediate state and the remaining 2% is transferred by Glu-165 to C2 of the same substrate molecule. This intramolecular transfer of tritium is therefore predicted to be independent of DHAP concentration. On the basis of NMR detection of a strong hydrogen bond between Glu-165 and the 1-OH of an analogue of the enediol intermediate [Harris, T. K., Abeygunawardana, C., and Mildvan, A. S. (1997) Biochemistry 36, 14661], we have suggested a "criss-cross" mechanism for TIM in which Glu-165 transfers a proton from C1 of DHAP to O2 of the enediol, and subsequently from O1 of the enediol to C2 of the product GAP. Since the pro-R proton is transferred to O2 instead of C2 in the criss-cross mechanism, no intramolecular transfer of label from substrate to product would be expected to occur. However, intermolecular transfer of label could occur if the label exchanges from O2 into a group on the protein and is transferred to GAP in subsequent turnovers. The extent of intermolecular tritium transfer in the criss-cross mechanism would be predicted to be dependent on DHAP concentration. The extent of tritium transfer was studied as a function of initial DHAP concentration using DHAP highly tritiated at the pro-R position. At 50% conversion to GAP, triphasic tritium transfer behavior was found. For phase 1, between 0.03 and 0.3 mM DHAP, a constant extent of tritium transfer of 1.19 +/- 0.03% occurred. For phase 2, between 0.3 and 1.0 mM DHAP, the extent of transfer progressively increased as a function of DHAP concentration to 2.17 +/- 0.15%. For phase 3, between 1.0 and 7.0 mM DHAP, the extent of transfer slightly decreased to 1.68 +/- 0.17%. In a direct test for intermolecular isotope transfer, doubly labeled [1(R)-D, 13C3]DHAP and 13C-depleted [1(R)-H,12C3]DHAP were synthesized, mixed in equal amounts, and incubated at 1 mM total DHAP with TIM, GAP dehydrogenase, NAD+, and arsenate until 50% conversion to 3-phosphoglycerate occurred. Electrospray ionization mass spectral analysis of the stable 3-phosphoglycerate product detected an extent of 1.4 +/- 0.4% of intramolecular D transfer from [13C3]DHAP to the 13C3 product, but no intermolecular transfer (

Distribution of carbohydrate epitopes among disjoint subsets of leech sensory afferent neurons.

Carbohydrate recognition plays an important role in the development of normal projections of sensory afferent neurons in the leech CNS. Four different carbohydrate epitopes are expressed by sensory afferents on their 130 kDa surface proteins: all sensory afferents share a common carbohydrate epitope (CE0) that helps them to enter and project diffusely across the synaptic neuropil; a restricted expression of three other carbohydrate epitopes (CE1, CE2, and CE3) serves to distinguish three subsets of sensory afferents. We examined the subsets of sensory afferents defined by their subset carbohydrate epitopes in the leech lip, skin, gut, and CNS. We established that the CE1, CE2, and CE3 subset epitopes define disjoint subsets of neurons by double labeling sensory afferents with monoclonal antibodies for different pairs of subset epitopes. We found that CE2 and CE3 afferents populate the lip and skin, but not the gut, and that these two subsets of sensory afferents have convergent projection patterns in the CNS. We found that CE1 afferents populate the gut and skin, but not lips; furthermore, their CNS projections diverge from those of CE2 and CE3 afferents. Our data fit the hypothesis that these carbohydrate epitopes are related to sensory modality of afferent subsets.

Detection and analysis of proteins modified by O-linked N-acetylglucosamine.

First, a protocol for increasing the stoichiometry of O-GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O-GlcNAc-modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O-linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O-GlcNAc modified proteins, including labeling of O-GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O-GlcNAc transferase and O-GlcNAcase activity, respectively.

Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta.

Estrogen receptor beta, a homologue to estrogen receptor alpha, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor alpha, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor beta is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor beta (mER-beta) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-beta, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-beta, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-beta from Sf9 cells is Ser(16) near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-beta at Ser(16). MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-beta suggests that these modifications may play a role in regulating estrogen receptor beta transactivation and turnover.