Homomultimeric Complexes Research Articles

Comprehensive proteomic characterization and functional annotation of Siberian sturgeon seminal plasma proteins

Siberian sturgeon (Acipenser baerii) is considered the most valuable species in aquaculture. The uniqueness of sturgeon reproduction implicates the need for studying semen composition at the protein level. Seminal plasma supports and protects the viability, motility and fertilizing capacity of spermatozoa by creating the optimal environment for storage and might be a rich source of biomarkers of reproductive physiology and/or disorders in male fishes. To our knowledge, there are no reports on the in-depth characterization of sturgeon seminal plasma proteins. The present study is the first to describe the in-depth proteomic characterization of Siberian sturgeon seminal plasma using two complementary proteomic approaches: gel-based (2DE and 2D blue native (BN)/SDS–PAGE) and gel-free (LC–MS/MS). LC–MS/MS allowed the identification of 657 proteins and 339 protein spots were identified after 2DE (almost 60% were present in various proteoforms). Combining the two proteomic strategies, we identified 665 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from sturgeon seminal plasma to date. For the first time, we demonstrated interactions between seminal plasma proteins; four multiprotein complexes (MC1-MC4) were identified after 2D BN/SDS–PAGE, including (MC1) serotransferrin-2 (TF) and fish-egg lectin (FEL); (MC2) serum albumin 2 (ALB) and retinol-binding protein 4 (RBP4); (MC3) ALB and TF; and (MC4) apolipoprotein A-I (APOA1), riboflavin-binding protein (RfBP) and myoglobin (MB). BN/SDS–PAGE also revealed that immunoglobulin and TF formed homomultimeric (dimer, trimer, tetramer and pentamer) complexes in seminal plasma. ALB, TF, beta-Ala-His dipeptidase (CNDP1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IGH, APOA1, hemopexin (HPX), type-4 ice-structuring protein (AFP4), FEL, RfBP, ovochymase 2 (OVCH2) and glycogen phosphorylase, liver form-like (PYGL) were selected as major seminal plasma proteins. The functional analysis of the identified proteins indicated their involvement mainly in immune system processes and response to stimulus, metabolism, vesicle-mediated transport, proteolysis, and cadherin binding involved in cell–cell adhesion. Moreover, 69 proteins were associated with reproductive processes, including spermatogenesis, fertilization, acrosomal reaction and sperm motility. Comparative proteomic analysis of sturgeon seminal plasma showed common proteins between fish and humans and 172 proteins specific for sturgeons, which may reflect the specificity of sturgeon reproduction. To conclude, this integrative proteomic view offers a deeper insight into the physiological function of seminal plasma and processes occurring in the sturgeon reproductive tract. The availability of a catalogue of sturgeon seminal plasma proteins opens the way to future research into development of new biomarkers and possible application in the improvement of methods of controlled reproduction. The mass spectrometry proteomics data have been deposited into ProteomeXchange with the identifier PXD035351.

Graph identification of proteins in tomograms (GRIP-Tomo).

In this study, we present a method of pattern mining based on network theory that enables the identification of protein structures or complexes from synthetic volume densities, without the knowledge of predefined templates or human biases for refinement. We hypothesized that the topological connectivity of protein structures is invariant, and they are distinctive for the purpose of protein identification from distorted data presented in volume densities. Three-dimensional densities of a protein or a complex from simulated tomographic volumes were transformed into mathematical graphs as observables. We systematically introduced data distortion or defects such as missing fullness of data, the tumbling effect, and the missing wedge effect into the simulated volumes, and varied the distance cutoffs in pixels to capture the varying connectivity between the density cluster centroids in the presence of defects. A similarity score between the graphs from the simulated volumes and the graphs transformed from the physical protein structures in point data was calculated by comparing their network theory order parameters including node degrees, betweenness centrality, and graph densities. By capturing the essential topological features defining the heterogeneous morphologies of a network, we were able to accurately identify proteins and homo-multimeric complexes from 10 topologically distinctive samples without realistic noise added. Our approach empowers future developments of tomogram processing by providing pattern mining with interpretability, to enable the classification of single-domain protein native topologies as well as distinct single-domain proteins from multimeric complexes within noisy volumes.

DNCON2_Inter: predicting interchain contacts for homodimeric and homomultimeric protein complexes using multiple sequence alignments of monomers and deep learning

Deep learning methods that achieved great success in predicting intrachain residue-residue contacts have been applied to predict interchain contacts between proteins. However, these methods require multiple sequence alignments (MSAs) of a pair of interacting proteins (dimers) as input, which are often difficult to obtain because there are not many known protein complexes available to generate MSAs of sufficient depth for a pair of proteins. In recognizing that multiple sequence alignments of a monomer that forms homomultimers contain the co-evolutionary signals of both intrachain and interchain residue pairs in contact, we applied DNCON2 (a deep learning-based protein intrachain residue-residue contact predictor) to predict both intrachain and interchain contacts for homomultimers using multiple sequence alignment (MSA) and other co-evolutionary features of a single monomer followed by discrimination of interchain and intrachain contacts according to the tertiary structure of the monomer. We name this tool DNCON2_Inter. Allowing true-positive predictions within two residue shifts, the best average precision was obtained for the Top-L/10 predictions of 22.9% for homodimers and 17.0% for higher-order homomultimers. In some instances, especially where interchain contact densities are high, DNCON2_Inter predicted interchain contacts with 100% precision. We also developed Con_Complex, a complex structure reconstruction tool that uses predicted contacts to produce the structure of the complex. Using Con_Complex, we show that the predicted contacts can be used to accurately construct the structure of some complexes. Our experiment demonstrates that monomeric multiple sequence alignments can be used with deep learning to predict interchain contacts of homomeric proteins.

Bacterial secretins: Mechanisms of assembly and membrane targeting.

Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.

Gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry: bovine insulin and ubiquitin.

Ion mobility-mass spectrometry (IMMS) is a very attractive method for studies in structural biology because of the ability of rapid isolation by nearly simultaneous m/z characterization and size separation, leading to an emergence of IMMS as a complimentary biochemical tool. Earlier, we developed a method based on varying the protein concentration in solution prior to electrospray ionization (ESI) with subsequent m/z selection and dissociation of protein multimers by IMMS of cytochrome c. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z for well-studied proteins bovine ubiquitin and insulin. These proteins were chosen due to their large difference in solution phase structures: insulin tightly bound by disulfide linkages, and ubiquitin-a protein that may adopt a range of states from compact to extended. Our preliminary results, as with cytochrome c reveal false negatives for protein oligomer formation and false positives for protein conformational states. In addition, these results will be couched in terms of the need for quantification of IMMS analysis of proteins given the total area under IMMS peaks can also distinguish conformation versus aggregation as higher order oligomers have more mass per ion.This article is part of the themed issue 'Quantitative mass spectrometry'.

Identification of tomoregulin-1 as a novel addicsin-associated factor

Addicsin is a novel factor encoding a 23-kDa hydrophobic protein that is highly upregulated in the amygdala nuclei of morphine-administered mice. It is a murine homolog of human JWA and rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a negative modulator of the neural glutamate transporter excitatory amino acid carrier 1 (EAAC1). Recent findings demonstrated that addicsin participates in various physiological processes by forming hetero- or homomultimeric complexes. However, the binding targets and molecular functions of addicsin remain largely unknown. To identify potential factors that associate with mouse addicsin, we performed a yeast two-hybrid screen using a 17-day-old mouse whole embryo cDNA library. We identified tomoregulin-1 (TR1) as a novel addicsin-associated factor. TR1, a type I transmembrane protein containing two follistatin-like modules and an epidermal growth factor-like domain, participates in nodal and bone morphogenetic protein signaling. Immunoprecipitation assays demonstrated that TR1 bound to addicsin, and that amino acids 145–188 of addicsin and amino acids 228–266 of TR1 were important for the formation of the addicsin-TR1 heterocomplex. The double-fluorescent immunohistochemical analysis revealed that addicsin and TR1 were coexpressed in neurons in the mature mouse brain regions tested. Moreover, TR1 showed a punctuate pattern throughout the cell, with preferential expression on the cell surface when expressed alone. However, TR1 predominantly redistributed to the endoplasmic reticulum (ER) when coexpressed with addicsin. Furthermore, coexpression of an addicsin mutant that lacked TR1 binding ability had little effect on the distribution of TR1. Biotinylation assays showed that coexpression of addicsin with TR1 suppressed the cell surface expression of TR1. Wound-healing assays demonstrated that the interaction of addicsin with TR1 had a significant effect on cell migration. These findings demonstrate that addicsin in the ER controls intracellular TR1 trafficking from the ER to plasma membrane and regulates cell migration through its interaction with TR1.

Critical Examination of Gas-Phase Protein Conformation/Multimer Ion Formation by Electrospray Ion Mobility-Mass Spectrometry

The generally accepted view of protein structure in the gas-phase is that protein ions produced by electrospray ionization (ESI) exist in a number of different states, and the resulting charge state distribution (CSD) and ion mobility spectrum is interpreted as evidence for protein ions retaining some memory of solution-phase conformation. Even with the inclusion of ion mobility information, reports of protein ion structure in the gas-phase are oftentimes in disagreement not only within the discipline but also as interpreted by other gas-phase techniques. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z. The concentration of cytochrome c in solution was varied over a wide range, and the multiply charged multimers (MCMs) present in the ion mobility/mass spectrum were unambiguously assigned by m/z selection and dissociation prior to ion mobility/mass spectrometry analysis. The results revealed false negatives for protein oligomer formation and false positives for protein conformational states and no evidence that gas-phase cytochrome c ions retain memory of solution-phase conformation, characteristics of great importance for structural biology. The results also suggest that the total IM-MS distribution for a protein is the complex result of individual MCMs either surviving until detection (undissociated) or dissociating into lower order multimers or a number of product ions for each m/z.

Abstract 1305: Fulvestrant induces the expression/activity of UGT1A4 in HepG2 cells: potential interactions with other therapeutic agents

Abstract Fulvestrant (Faslodex™) is a pure antiestrogen that has been shown to be effective in treating estrogen receptor (ER) positive tumors that are resistant to selective estrogen receptor modulators (SERMs) such as tamoxifen. Currently, clinical trials investigating the utility of adding fulvestrant to other therapeutics are being conducted. While co-administration of these drugs has not been shown to affect cytochrome P450-mediated metabolism of either drug, effects of co-administration on Phase II metabolism (i.e. glucuronidation) have not been explored. We demonstrated that fulvestrant up-regulates expression of UDP glucuronosyltransferase (UGT) 1A4, the major enzyme responsible for the direct conjugation and subsequent elimination of many therapeutic drugs. Endogenous mRNA expression levels of UGT1A4 in HepG2 cells were induced 4.5-fold by fulvestrant treatment in this ERα-expressing cell line. Treatment with fulvestrant upregulated UGT1A4 in a time- (0-72 hours) and concentration-(0-250 nM) dependent manner and silencing ERα with specific siRNA abolished UGT1A4 up regulation. The role of a Sp1 binding site (−1906 to -1901 base pairs), known to be critical for the induction of UGT1A4 transcriptional activity by estradiol, was examined in relation to fulvestrant-mediated induction of UGT1A4. Electrophoretic mobility shift assays were performed using these nuclear extracts and UGT1A4/Sp1 probe. Fulvestrant-treated HepG2 nuclear extract formed multiple shifted bands that appear to represent homomultimeric complexes of Sp1 with UGT1A4/Sp1 probe. This binding ability was partially blocked by mithramycin, a known inhibitor of Sp1 binding to DNA. While these in vitro assays indicate that fulvestrant administration could influence the disposition of other UGT1A4 substrates, the clinical significance of these findings is unclear. Further studies are needed to assess the effects of fulvestrant administration on the disposition of drugs that are substrates of UGT1A4, including anastrozole, clozapine, imipramine, irinotecanl, amotrigine, meperidine, midazolam, nicotine, olanzapine, SN-38 and tamoxifen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2011-1305

Potential in targeting classical inward rectifier channels

t c Inward rectifier channels were first described in skeletal myocytes as constitutively open potassium-selective channels conducting potassium ions more efficiently in the inward than the outward direction. Later, it was realized that currents with these characteristics are present in many organs, including brain and heart. The diode-like behavior of these channels is based on internal block by Mg and/or polyamines and is crucial for the diverse functions in cell physiologic contexts. Classic inwardly rectifying currents are conducted by a family of Kir2.x proteins. Currently, six Kir2 subunits (Kir2.1–2.6) were described that can assemble to form homomultimeric or heteromultimeric channel complexes. Kir2.1/2.2/2.3 heteromers were suggested to be the structural basis of cardiac channels conducting IK1. IK1 significantly contributes to repolarizing potassium conductance in cardiac late-phase repolarization and stabilizes the resting membrane potential of atrial and ventricular myocytes. Several clinical observations support the concept of an antiarrhythmic effect by Kir2.x modulation and, consequently, small molecule modulators acting on inwardly rectifying K channels were reported as potential promising antiarrhythmic drugs. Kir2.x channel expression and subunit composition change during diverse forms of arrhythmia. Furthermore, overexpression of active or dominant-negative Kir2 channel variants increases the susceptibility of diverse forms of ventricular or atrial arrhythmia, as summarized by van der Heyden and Sanchez-Chapula in their study reported in this issue of eart Rhythm. These data indicate that inwardly rectifying K channels play a key role during development and maintenance of arrhythmias. Therefore, modulation of these channels holds the potential to counteract arrhythmia susceptibility as well. As already described for other antiarrhythmic drugs, inward rectifier targeting compounds most probably will have both proarrhythmic and antiarrhythmic potential. An obvious additional hurdle to overcome in the development of Kir2.x-based antiarrhythmics will be avoiding teratogenic effects, which are suggested by observed impairments such as syndactyly and facial dysmorphia caused by Kir2.1 loss-of-function mutations in Andersen-Tawil syndrome. Therefore, use of these currently

Identification of cytoplasmic and membrane-associated complexes in human embryonic stem cells using blue native PAGE

Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.

Organellar Protein Complexes of Caco-2 Human Cells Analyzed by Two-Dimensional Blue Native/SDS-PAGE and Mass Spectrometry

The complexome is essential for a better understanding of protein functions. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. Two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) technology is thus an interesting and powerful approach for this purpose. This report deals with the analysis and the identification of the organellar protein complexes of Caco-2 human cells using 2D BN/SDS-PAGE and HPLC-chip-MS. We identified 58 protein complexes (26 heteromultimeric and 32 homomultimeric complexes) and 4 monomeric proteins. Among them, 32 protein complexes were pointed out, providing insights into the function of previously uncharacterized human proteins.

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

The 29-Nucleotide Deletion Present in Human but Not in Animal Severe Acute Respiratory Syndrome Coronaviruses Disrupts the Functional Expression of Open Reading Frame 8

One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab(+)), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.

The role of CD44 splice variants in human metastatic cancer.

The large family of CD44 splice variants are likely to serve multiple functions in the embryo and in the adult organism. This is reflected in their complex patterns of expression. In molecular terms these functions are largely unknown. Certain splice variants (CD44v) can promote the metastatic behaviour of cancer cells. In human colon and breast cancer the presence of epitopes encoded by exon v6 on primary resected tumour material indicates poor prognosis. Metastasis-promoting splice variants differ from those that seem not to have a role in the induction of metastasis by the formation of homomultimeric complexes in the plasma membrane of cells. This may increase their affinity to ligands such as hyaluronate. The affinity can be further regulated over a range from low to very high by cell-specific modification. The fact that CD44v epitopes are found on normal epithelial cells such as skin, cervical epithelium and bladder enforces cautious evaluation of the significance of CD44v expression in human cancer. Nevertheless, certain epitopes can serve as tools in early diagnosis of certain cancers and will facilitate the development of specific targeted therapy.

TatBC, TatB, and TatC form structurally autonomous units within the twin arginine protein transport system of Escherichia coli

The Tat (twin arginine translocation) system transports folded proteins across bacterial and thylakoid membranes. The integral membrane proteins TatA, TatB, and TatC are the essential components of the Tat pathway in Escherichia coli. We demonstrate that formation of a stable complex between TatB and TatC does not require TatA or other Tat components. We show that the TatB and TatC proteins are each able to a form stable, defined, homomultimeric complexes. These we suggest correspond to structural subcomplexes within the parental TatBC complex. We infer that TatC forms a core to the TatBC complex on to which TatB assembles.

Elucidating Factors Manipulated the Formation of Multiply Charged Protein Homomultimeric Complexes by Electrospray Ionization

AbstractNative non‐covalently bonded protein‐protein and protein‐substrate complexes are of great interest and have been extensively studied by electrospray ionization mass spectrometry (ESI‐MS). Multiply charged protein homomultimeric complexes are shown to form by ESI‐MS. This study addresses factors that can artificially induce the formation of multiply charged protein homomultimeric complexes. Cytochrome c (Cyt c) and ubiquitin, which are monomers in solution, were found to generate (Cyt c)mn+ by electrospray ionization (ESI). The homomultimeric complexes were not limited to dimeric complexes but include also multiply charged trimers, tetramers, and pentamers. The observation of these homomultimeric complexes has never been revealed from a Cyt c solution at the concentration as low as 10 μM. Increasing the concentration of Cyt c enhanced the formation of (Cyt c)mn+ as expected; however, the protein concentration does not affect the relative intensities of monomeric and dimeric complexes. Additionally the enrichment of NH4OH also promotes the formation of (Cyt c)mn+. Notably, source collision‐induced dissociations (source‐CID) of (Cyt c)mn+ alter the charge state distribution (CSD) and may lead to an incorrect interpretation of Cyt c conformations. Hence, extra care should be taken when using CSD to interpret the conformation of a protein derived from ESI‐MS.

The TatA d component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatA d was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatA d formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatA d independent from TatC d, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatA d complexes.

Novel Myocilin Mutation in a Chinese Family With Juvenile-Onset Open-Angle Glaucoma

To search for the genetic cause of juvenile-onset open-angle glaucoma (JOAG) in a Chinese family. In a 3-generation glaucoma family affected with JOAG or ocular hypertension, we screened myocilin (MYOC) and optineurin (OPTN) for mutations and investigated apolipoprotein E (APOE) polymorphisms in 6 family members, 2 of them patients with JOAG, 2 patients with ocular hypertension, and 2 patients who were asymptomatic. Normal controls included 200 unrelated Chinese subjects. The COS-7 cell line was transfected with expression vectors encoding wild-type or mutated MYOC complementary DNA. Cellular and secreted MYOC proteins were characterized by Western blotting. One missense MYOC mutation, 734G>A: Cys245Tyr, was identified. It occurred in all 4 family members afflicted with JOAG or ocular hypertension but not in asymptomatic family members. No OPTN variations were observed. APOE polymorphism frequencies were similar to those for controls. The Cys245Tyr MYOC mutation cosegregated with the disorder within the family. It was absent in the 200 control subjects. The Cys245Tyr mutant MYOC protein formed homomultimeric complexes that migrated at molecular weights larger than their wild-type counterparts. These mutant complexes remained sequestered intracellularly in COS-7 cells. The Cys245Tyr MYOC mutation was the genetic cause of JOAG in this Chinese family. This mutation may alter covalent bonds that formed between MYOC cysteines. Clinical Relevance Genetic tests of MYOC mutations may be beneficial to predict new cases of the disease in families with JOAG.

Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking

CD22 is a negative regulator of B-cell receptor signaling, an activity mediated by recruitment of SH2 domain-containing phosphatase 1 through a phosphorylated immunoreceptor tyrosine inhibitory motif in its cytoplasmic domain. As in other members of the sialic acid-binding immunoglobulin-like lectin, or siglec, family, the extracellular N-terminal immunoglobulin domain of CD22 binds to glycan ligands containing sialic acid, which are highly expressed on B-cell glycoproteins. B-cell glycoproteins bind to CD22 in cis and 'mask' the ligand-binding domain, modulating its activity as a regulator of B-cell signaling. To assess cell-surface cis ligand interactions, we developed a new method for in situ photoaffinity cross-linking of glycan ligands to CD22. Notably, CD45, surfaceIgM (sIgM) and other glycoproteins that bind to CD22 in vitro do not appear to be important cis ligands of CD22 in situ. Instead, CD22 seems to recognize glycans of neighboring CD22 molecules as cis ligands, forming homomultimeric complexes.