Isolation and characterization of a novel IgD-binding protein from Moraxella catarrhalis

A novel surface protein of the bacterial species Moraxella catarrhalis that displays a high affinity for IgD (MID) was solubilized in Empigen and isolated by ion exchange chromatography and gel filtration. The apparent molecular mass of monomeric MID was estimated to approximately 200 kDa by SDS-PAGE. The mid gene was cloned and expressed in Escherichia coli. The complete mid nucleotide gene sequence was determined, and the deduced amino acid sequence consists of 2123 residues. The sequence of MID has no similarity to other Ig-binding proteins and differs from all previously described outer membrane proteins of M. catarrhalis. MID was found to exhibit unique Ig-binding properties. Thus, in ELISA, dot blots, and Western blots, MID bound two purified IgD myeloma proteins, four IgD myeloma sera, and finally one IgD standard serum. No binding of MID was detected to IgG, IgM, IgA, or IgE myeloma proteins. MID also bound to the surface-expressed B cell receptor IgD, but not to other membrane molecules on human PBLs. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunological research.     

Functional mapping of an oligomeric autotransporter adhesin of Aggregatibacter actinomycetemcomitans

Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.     

Organization of the AAA(+) adaptor protein PspA is an oligomeric ring

The 25.3 kDa "adaptor" protein, PspA (phage shock protein A), is found in the cytoplasm and in association with the inner membrane of certain bacteria. PspA plays critical roles in negatively regulating the phage shock response and maintaining membrane integrity, especially during the export of proteins such as virulence factors. Homologues of PspA function exist for thylakoid biogenesis. Here we report the first three-dimensional reconstruction of a PspA assembly from Escherichia coli, visualized by electron microscopy and single particle analysis to a resolution of 30 Angstroms. The assembly forms a 9-fold rotationally symmetric ring with an outer diameter of 200 Angstroms, an inner diameter of 95 Angstroms, and a height of approximately 85 Angstroms. The molecular mass of the complex was calculated to be 1023 kDa by size exclusion chromatography, suggesting that each of the nine domains is likely to be composed of four PspA subunits. The functional implications of this PspA structure are discussed in terms of its interaction with the protein export machinery of the bacterial cell and its AAA(+) protein partner, PspF.     

The immunoglobulin D-binding part of the outer membrane protein MID from Moraxella catarrhalis comprises 238 amino acids and a tetrameric structure

Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID(962-1200)). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID(985-1142)) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID(962-1200) was efficiently attracted to a standard IgD serum and to purified myeloma IgD(kappa) and IgD(lambda) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID(962-1200) using transposons suggested that alpha-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID(962-1200) was a tetramer. Interestingly, tetrameric MID(962-1200) attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID(962-1200) is crucial for optimal IgD-binding capacity.     

Human immunodeficiency virus type 1 Vpu protein is an oligomeric type I integral membrane protein.

The human immunodeficiency virus type 1 Vpu protein is a 16-kDa phosphoprotein which enhances the efficiency of virion production and induces rapid degradation of CD4, the cellular receptor for human immunodeficiency virus. The topology of membrane-inserted Vpu was investigated by using in vitro-synthesized Vpu cotranslationally inserted into canine microsomal membranes. Proteolytic digestion and immunoprecipitation studies revealed that Vpu was a type I integral membrane protein, with the hydrophilic domain projecting from the cytoplasmic membrane face. In addition, several high-molecular-weight proteins containing Vpu were identified by chemical cross-linking. Such complexes also formed when wild-type Vpu and a Tat-Vpu fusion protein were coexpressed. Subsequent analysis by one- and two-dimensional electrophoresis revealed that these high-molecular-weight complexes consisted of homo-oligomers of Vpu. These findings indicate that Vpu is a type I integral membrane protein capable of multimerization.     

The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated

Nontypeable Haemophilus influenzae is a gram-negative commensal organism that is commonly associated with localized respiratory tract disease. The pathogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells. Hia is the major adhesin expressed by a subset of nontypeable H. influenzae strains and promotes efficient adherence to a variety of human epithelial cell lines. Based on previous work, Hia is transported to the surface of Escherichia coli transformants and is capable of mediating E. coli adherence without the assistance of other H. influenzae proteins. In the present study, we examined the mechanism of Hia secretion. PhoA fusions, deletional mutagenesis, and N-terminal amino acid sequencing established that the signal for Hia export from the cytoplasm resides in the first 49 amino acids, including a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelectron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure analysis predicted that the C terminus of Hia forms a beta-barrel with a central hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter proteins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hia is the prototype of an important subfamily of autotransporter proteins.     

An oligomeric protein is imported into peroxisomes in vivo

The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co- expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA- CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.     

The autotransporter protein from Bordetella avium, Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

The autotransporter protein from Bordetella avium,Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.     

Na/K-ATPase as an oligomeric ensemble

Differences in the kinetic behavior and properties of monomeric and oligomeric forms of membrane-bound Na/K-ATPase are analyzed. It is concluded that enzyme molecules within oligomeric complexes are affected by extrinsic signals that result in change of enzyme activity, whereas the individual (protomeric) state is insensitive to these signals. Some of the major factors of such regulation are microviscosity of the lipid environment, reactive oxygen species, and intracellular protein kinases.     

The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1-17 and an unknown number of residues between 17-28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8-13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 of Drosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.     

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram-negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti-App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.     

Vectorial transport and folding of an autotransporter virulence protein during outer membrane secretion

Autotransporter (AT) proteins are a large and diverse family of extracellular virulence proteins from Gram-negative bacteria, characterized by a central β-helix domain within the mature virulence protein. It is not clear how these proteins cross the outer membrane (OM) quickly and efficiently, without assistance from an external energy source such as ATP or a proton gradient. Conflicting results in the literature have led to several proposed mechanisms for AT OM secretion, including a concerted process, or vectorial secretion with different directionalities. We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis , and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain β-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.     

Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.     

Small heat shock protein activity is regulated by variable oligomeric substructure

The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified alpha-crystallins, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the alpha-crystallins in protein deposition diseases.