Phosphatase-like Domain Research Articles

Structure–function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

New insights into regulation of P‐Rex1 autoinhibition

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)–dependent Rac exchanger 1 (P‐Rex1) is a guanine nucleotide exchange factor for Rho GTPases downstream of G protein coupled receptors (GPCRs) and tyrosine kinases. It is primarily expressed in hematopoietic cells and brain. Its ectopic expression is reported in different cancers including breast, prostrate, skin, and ovarian cancers, making it a potential therapeutic target. P‐Rex1 is a multi‐domain protein consisting of a catalytic Dbl‐homology (DH) domain, followed by a PH, two DEP, two PDZ, and an inositolpolyphosphate‐4 phosphatase‐like (IP4P) domain. P‐Rex1 is regulated by PIP3, Gbg binding and phosphorylation via PKC and PKA. In unstimulated cells, P‐Rex1 is autoinhibited by intradomain interactions with its catalytic DH domain. Previously, our lab had shown that first DEP (DEP1) domain plays a key role in inhibition of DH/PH‐DEP1 exchange activity. However, there is no information available as how these domains are engaged in regulation of full length P‐Rex1. In our current study, we addressed this gap by employing single particle cryo‐electron microscopy (cryo‐EM) complemented by SAXS, HDX‐MS, and functional enzymatic assays to understand P‐Rex1 regulation. A structure with the head group of PIP3 (IP4) bound to the PH domain revealed interactions between DH and DEP1 domain that would block GTPase access to DH domain by coercing a novel configuration of the DH‐PH module. In addition, we observed that the IP4P domain was anchored to PH domain via a previously unrecognized FAT domain. Mutagenesis of residues involved in the DH–DEP1 interface led to activation, suggestive of inhibitory role of this interface. IP4 is a soluble second messenger signaling molecule which is reported to compete with PIP3 binding site in other proteins. Therefore, we tested whether it could regulate P‐Rex1 in a similar manner and found that IP4 competes with PIP3 and negatively regulates P‐Rex1 activity. Based on these observations, we propose that in unstimulated cell, P‐Rex1 is autoinhibited by intradomain interactions between the DEP1 and DH and between the FAT and PH domains, and that inositol phosphate levels may add an additional layer of desensitization by opposing P‐Rex1 membrane translocation and favoring a closed autoinhibited state.

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD.

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.

The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Mechanisms of PTPσ-Mediated Presynaptic Differentiation.

Formation of synapses between neurons depends in part on binding between axonal and dendritic cell surface synaptic organizing proteins, which recruit components of the developing presynaptic and postsynaptic specializations. One of these presynaptic organizing molecules is protein tyrosine phosphatase σ (PTPσ). Although the protein domains involved in adhesion between PTPσ and its postsynaptic binding partners are known, the mechanisms by which it signals into the presynaptic neuron to recruit synaptic vesicles and other necessary components for regulated transmitter release are not well understood. One attractive candidate to mediate this function is liprin-α, a scaffolding protein with well-established roles at the synapse. We systematically mutated residues of the PTPσ intracellular region (ICR) and used the yeast dihydrofolate reductase (DHFR) protein complementation assay to screen for disrupted interactions between these mutant forms of PTPσ and its various binding partners. Using a molecular replacement strategy, we show that disrupting the interaction between PTPσ and liprin-α, but not between PTPσ and itself or another binding partner, caskin, abolishes presynaptic differentiation. Furthermore, phosphatase activity of PTPσ and binding to extracellular heparan sulfate (HS) proteoglycans are dispensable for presynaptic induction. Previous reports have suggested that binding between PTPσ and liprin-α is mediated by the PTPσ membrane-distal phosphatase-like domain. However, we provide evidence here that both of the PTPσ phosphatase-like domains mediate binding to liprin-α and are required for PTPσ-mediated presynaptic differentiation. These findings further our understanding of the mechanistic basis by which PTPσ acts as a presynaptic organizer.

Cloning and expression of the carbaryl hydrolase gene mcbA and the identification of a key amino acid necessary for carbaryl hydrolysis

Carbamate hydrolase is the initial and key enzyme for degradation of carbamate pesticides. In the present study, we report the isolation of a carbaryl-degrading strain Pseudomonas sp. XWY-1, the cloning of its carbaryl hydrolase gene (mcbA) and the characterization of McbA. Strain XWY-1 was able to utilize carbaryl as a sole carbon source and degrade it using 1-naphthol as an intermediate. Transposon mutagenesis identified a mutant of XWY-1M that was unable to hydrolyze carbaryl. The transposon-disrupted gene mcbA was cloned by self-formed adaptor PCR, then expressed in Escherichia coli BL21(DE3) and purified. McbA was able to hydrolyze carbamate pesticides including carbaryl, isoprocarb, fenobucarb, carbofuran efficiently, while it hydrolyzed aldicarb, and propoxur poorly. The optimal pH of McbA was 7.0 and the optimal temperature was 40°C. The apparent Km and kcat values of McbA for carbaryl were 77.67±12.31μM and 2.12±0.10s−1, respectively. Three amino acid residues (His467, His477 and His504) in the predicted polymerase/histidinol phosphatase-like domain were shown to be closely related to the activity of McbA, with His504 being the most important, as a replacement of His504 led to the complete loss of activity. This is the first study to identify key amino acids in McbA.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

Landes Highlights

MiR-34a inhibits GBM growth by targeting notch1RNA as a structural organizer of chromatinNovel RNAP II binding partners identified: RPRD proteinsIdentification and modeling of a phosphatase-like domain in a tRNA 2′-O-ribosyl phosphate transferase Rit1pTumor-suppressive miR-9 silenced via promoter hypermethylation

Identification and modeling of a phosphatase-like domain in a tRNA 2′-O-ribosyl phosphate transferase Rit1p

Cytoplasmic initiator tRNAs from plants and fungi are excluded from participating in translational elongation by the presence of a unique 2'-phosphoribosyl modification of purine 64, introduced posttranscriptionally by the enzyme Rit1p. Members of the Rit1p family show no obvious similarity to other proteins or domains, there is no structural information available to guide experimental analyses, and the mechanism of action of this enzyme remains a mystery. Using protein fold recognition, we identified a phosphatase-like domain in the C-terminal part of Rit1p. A comparative model of the C-terminal domain was constructed and used to predict the function of conserved residues and to propose the mechanism of action of Rit1p. The model will facilitate experimental analyses of Rit1p and its interactions with the initiator tRNA substrate.

막 전위 감지 탈인산화 효소, Ci-VSP의 유사 탈인산화 효소 도메인의 발현과 정제

최근에 Ciona intestinalis에서 확인된 막 전위 감지 탈인산화 효소는 이온 채널과 유사한 막 통과 도메인과 유사 탈인산화 효소 도메인으로 이루어져 있다. 이 Ci-VSP는 그 막 통과 도메인에 의해 막 전위 변화를 감지하고 유사 탈인산화 효소에 의해 인산화된 이노시틸 인지질들에 대한 탈인산화 활성을 보이는 것으로 알려져 있다. 본 연구는 6개의 히스티딘이 융합된 Ci-VSP의 유사 탈인산화 효소 도메인의 발현 시스템 구축을 추구하였다. 그래서 본 논문은 그 시스템의 구축과 발현 그리고 정제 조건 확립, 마지막으로 그 효소 동력학적인 활성을 pNPP에 대한 검토한 결과를 보고한다. 본 연구 결과에 따르면 Ci-VSP(248-576)-His 단백질 발현 및 정제시에 다른 단백질들의 정제 조건과 다르게 25 mM NaCl과 100 mM 정도의 이미다졸이 필요함을 확인하였다. 정제된 단백질을 가지고 탈인산화 효소의 대표적인 기질인 pNPP를 이용하여 그 동력학적인 상수 측정을 시도한 결과 정상 상태 동력학 측정 조건으로 온도 <TEX>$37^{\circ}C$</TEX>, pH 5.0 내지 5.5, 반응 시간 30분 이내, 반응 단백질 양 <TEX>$2.0\;{\mu}g$</TEX> 이내가 적합하다는 점을 알게 되었다. 이러한 확립된 조건을 토대로 pNPP에 대한 동력학적 상수를 측정한 결과 <TEX>$K_m$</TEX> 값은 <TEX>$354{\pm}0.143\;{\mu}M$</TEX>이며 <TEX>$V_{max}$</TEX>는 <TEX>$0.0607{\pm}0.0137\;{\mu}mol$</TEX>/min/mg이며 <TEX>$k_{cat}$</TEX> 값은 <TEX>$2.359{\pm}0.009751\;min^{-1}$</TEX>인 것으로 확인되었다. 이를 통해 본 연구는 Ci-VSP(248-576)-His의 순도 높은 정제 결과와 pNPP에 대한 높은 활성 보임을 제시하였다. 이러한 연구 결과를 통해 향후 Ci-VSP에 대한 구조적인 연구 등을 포함하는 다양한 생화학적인 연구가 수행되리라 본다. Recently identified Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) consists of an ion channel-like transmembrane domain (VSD) and a phosphatase-like domain. Ci-VSP senses the change of membrane potential by its VSD and works as a phosphoinositide phosphatase by its phosphatase domain. In this study, we present the construction of His-tagged phosphatase-like domain of Ci-VSP, its recombinant expression and purification, and its enzymatic activity behavior in order to examine the biochemical behavior of phosphatase domain of Ci-VSP without interference. We found that Ci-VSP(248-576)-His can be eluted with an elution buffer containing 25 mM NaCl and 100 mM imidazole during His-tag purification. In addition, we found the proper measurement condition for kinetics study of Ci-VSP(248-576)-His against p-nitrophenyl phosphate (pNPP). We measured the kinetic constant of Ci-VSP(248-576)-His at <TEX>$37^{\circ}C$</TEX>, pH 5.0 or 5.5, under 30 min of reaction time, and less than <TEX>$2.0\;{\mu}g$</TEX> of protein amount. With these conditions, we acquired that Ci-VSP(248-576)-His has <TEX>$K_m$</TEX> of <TEX>$354{\pm}0.143\;{\mu}M$</TEX>, <TEX>$V_{max}$</TEX> of <TEX>$0.0607{\pm}0.0137\;{\mu}mol$</TEX>/min/mg and <TEX>$k_{cat}$</TEX> of <TEX>$0.359{\pm}0.009751\;min^{-1}$</TEX> for pNPP dephosphorylation. Therefore, we produced a pure form of Ci-VSP(248-576)-His, and this showed a higher activity against pNPP. This purified protein will provide the road to a structural investigation on an interesting protein, Ci-VSP.

Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-β and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain

The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 K(d)=96 nM; PPIP5K2 K(d)=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

Autoantibodies against IA-2, GAD, and topoisomerase II in type 1 diabetic patients

Prevalence of autoantibodies against IA-2 (IA-2A), glutamic acid decarboxylase (GADA), and type II DNA topoisomerase (TopIIA) of Taiwanese type 1 diabetes mellitus (T1DM) patients was investigated. Correlations of these autoantibodies with patients’ clinical manifestations were also analyzed. Prevalence of IA-2A, GADA, and TopIIA in our patients was 23.6%, 47.1%, and 55.2%, respectively. Eighty percent of the IA-2A recognized the carboxyl terminus of the IA-2 protein tyrosine phosphatase-like domain. Average disease duration of IA-2A + patients was significantly shorter than that of IA-2A − patients [3.76 ± 0.42 vs. 4.98 ± 0.34 years, p=0.028]. Presence of GADA was correlated with the mean age of onset [10.82 ± 0.76 vs. 8.38 ± 0.77 years for GADA + and GADA − patients, p=0.026]. Patients with adolescent onset have higher GADA prevalence and better residual β-cell functions. TopIIA and GADA are suggested to be better markers for Taiwanese T1DM patients because of their higher prevalence and persistence.

The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of β2-syntrophin and nNOS in pancreatic β-cells

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

The PPS1 Gene of Saccharomyces cerevisiae Codes for a Dual Specificity Protein Phosphatase with a Role in the DNA Synthesis Phase of the Cell Cycle

We report the identification of the PPS1 gene of Saccharomyces cerevisiae. The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues. Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension. Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity. The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein. PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase. pps1Delta mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S. cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis. A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest. The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase.

Evaluation of islet cell antigen (ICA) 512/IA-2 autoantibody radioassays using overlapping ICA512/IA-2 constructs.

Islet cell antigen (ICA) 512 also termed IA-2 is a novel autoantigen of type 1 diabetes, which has a tyrosine phosphatase-like domain. We have assessed autoantibody RIAs using a series of ICA512/IA-2 constructs to produce in vitro synthesized 35S-methionine-labeled proteins. Levels of ICA512/IA-2 (256-979, truncated aminoterminus) autoantibodies were strongly correlated with those of the full-length ICA512/IA-2 (1-979) autoantibodies (r = 0.96, P < 0.0001) and ICA512/IA-2 (687-979) autoantibodies (r = 0.98, P < 0.0001). RIAs using these 3 constructs had increased sensitivity relative to our initially reported ICA512 autoantibody RIA (amino acids 389-948, truncated carboxy- and aminoterminus). Only 2 of 38 sera examined in this study reacted with an aminoterminus ICA512/IA-2 (1-577) construct. The mean SD score (SD score = (index of unknown sample-mean index of controls)/SD of controls) using the ICA512/IA-2 (256-979) construct was significantly higher than the SD score obtained with other ICA512/IA-2 constructs (P < 0.001). Amongst patients with new-onset diabetes and prediabetic relatives, using RIAs for autoantibodies reacting with ICA512/IA-2 (256-979), insulin, and glutamic acid decarboxylase 65, 98% expressed one or more of these autoantibodies and 78% expressed two or more, whereas no control (n = 208) expressed more than a single autoantibody. A combined ICA512/IA-2 (256-979), glutamic acid decarboxylase 65 autoantibody RIA with differential autoantigen labeling (35S-methionine, 3H-leucine) has been developed that uses 96-well plate membrane filtration and Top Counter beta counting. Concordance between results of dual and single RIAs was greater than 90%. This simple combined autoantibody assay should facilitate large-scale autoantibody screening.

Value of ICA512 antibodies for prediction and diagnosis of type 1 diabetes

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting β-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds β2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel β2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length β2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the β2-syntrophin PDZ domain to ICA512 required both ICA512′s C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-β2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain β2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through β2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

Autoantibodies in insulin-dependent diabetes recognize distinct cytoplasmic domains of the protein tyrosine phosphatase-like IA-2 autoantigen.

Protein tyrosine phosphatase-like IA-2 is a major autoantigen in insulin-dependent diabetes. It has been identified as both a specificity of cytoplasmic islet cell Abs and one of the precursors of the 40- and 37-kDa tryptic fragment islet autoantigens. To characterize autoantibody binding to IA-2 and determine whether humoral autoimmunity extends to other tyrosine phosphatases, we analyzed serum reactivity in 100 patients with insulin-dependent diabetes against different in vitro translated portions of the IA-2 protein as well as the tyrosine phosphatase domains of HPTPbeta and HPTPdelta. All autoantibody reactivity was confined to the cytoplasmic portion of IA-2 (amino acids 601-979). At least four epitopes were found. These were contained within amino acids 605 to 620 and 605 to 682 of the juxtamembrane region and within amino acids 777 to 937 and 687 to 979 in the IA-2 tyrosine phosphatase-like domain. Footprinting studies confirmed the presence of multiple epitopes. Fifty-six percent of sera with IA-2 Abs bound epitopes within the juxtamembrane region, and 83% bound epitopes in the tyrosine phosphatase-like domain; 39% had Abs to both regions. No reactivity against the IA-2 ectodomain or the tyrosine phosphatase domains of HPTPbeta and HPTPdelta was found. These data suggest that the cytoplasmic region, in particular the tyrosine phosphatase-like domain, is the major target of IA-2 Abs in insulin-dependent diabetes, and that autoantibody reactivity is specific for IA-2 or IA-2-like molecules.