Asp2 and Asp3 interact directly with GspB, the export substrate of the Streptococcus gordonii accessory Sec System

GspB is a serine-rich glycoprotein adhesin of Streptococcus gordonii that is exported to the bacterial surface by the accessory Sec system. This dedicated export pathway is comprised of seven components (SecA2, SecY2, and five accessory Sec proteins [Asp1 to Asp5]). The latter proteins have no known homologs beyond the Asps of other species. Asp1 to Asp3 are absolutely required for export of the substrate GspB, but their roles in this process are unknown. Using copurification analysis and far-Western blotting, we found that Asp2 and Asp3 could individually bind the serine-rich repeat (SRR) domains of GspB. Deletion of both SRR regions of GspB led to a decrease in its export, suggesting that binding of the Asps to the SRR regions is important for GspB transport by the accessory Sec system. The Asps also bound a heterologous substrate for the accessory Sec system containing a slow-folding MalE variant, but they did not bind wild-type MalE. The combined results indicate that the Asps may recognize the export substrate through preferential interactions with its unstructured or unfolded regions. Glycosylation of the SRR domains on GspB prevented Asp binding, suggesting that binding of the Asps to the preprotein occurs prior to its full glycosylation. Together, these findings suggest that Asp2 and Asp3 are likely to function in part as chaperones in the early phase of GspB transport.     

Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system

GspB is a large cell-surface glycoprotein expressed by Streptococcus gordonii M99 that mediates binding of this organism to human platelets. This adhesin is glycosylated in the cytoplasm, and is then transported to the cell surface via an accessory Sec system. To assess the structural features of GspB that are needed for export, we examined the effects of altering the carbohydrate moieties or the polypeptide backbone of GspB. Truncated, glycosylated variants of GspB were exported exclusively via the accessory Sec pathway. When glycosylation was abolished, the GspB variants were still exported by this pathway, but minor amounts could also be transported by the canonical Sec system. GspB variants with in-frame insertions or deletions in the N-terminus were not secreted, indicating that this domain is necessary for export. However, the N-terminus is not sufficient for the transport of heterologous proteins, because C-terminal fusion of passenger proteins to this domain hindered export. In contrast, fusion of GspB to a canonical signal peptide resulted in the efficient export of non-glycosylated forms of the fusion protein via the canonical Sec pathway, whereas glycosylated forms could not be exported. Thus, the carbohydrate moieties and the atypical signal sequence of GspB interfere with export via the canonical pathway, and direct GspB towards the accessory Sec system.     

Canonical SecA associates with an accessory secretory protein complex involved in biogenesis of a streptococcal serine-rich repeat glycoprotein

Fap1, a serine-rich repeat glycoprotein (SRRP), is required for bacterial biofilm formation of Streptococcus parasanguinis. Fap1-like SRRPs are found in many gram-positive bacteria and have been implicated in bacterial fitness and virulence. A conserved five-gene cluster, secY2-gap1-gap2-gap3-secA2, located immediately downstream of fap1, is required for Fap1 biogenesis. secA2, gap1, and gap3 encode three putative accessory Sec proteins. SecA2 mediates export of mature Fap1, and Gap1 and Gap3 are required for Fap1 biogenesis. Interestingly, gap1 and gap3 mutants exhibited the same phenotype as a secA2 mutant, implying that Gap1 and Gap3 may interact with SecA2 to mediate Fap1 biogenesis. Glutathione S-transferase pulldown experiments revealed a direct interaction between SecA2, Gap1, and Gap3 in vitro. Coimmunoprecipitation analysis demonstrated the formation of a SecA2-Gap1-Gap3 complex. Homologues of SecA2, Gap1, and Gap3 are conserved in many streptococci and staphylococci. The corresponding homologues from Streptococcus agalactiae also interacted with each other and formed a protein complex. Furthermore, the Gap1 homologues from S. agalactiae and Streptococcus sanguinis rescued the Fap1 defect in the Gap1 mutant, indicating the functional conservation of the accessory Sec complex. Importantly, canonical SecA interacted with the accessory Sec protein complex, suggesting that the biogenesis of SRRPs mediated by the accessory Sec system is linked to the canonical Sec system.     

Asp3 mediates multiple protein–protein interactions within the accessory Sec system of Streptococcus gordonii

Bacterial binding to human platelets is an important step in the pathogenesis of infective endocarditis. Streptococcus gordonii can mediate its platelet attachment through a cell wall glycoprotein termed GspB (‘gordonii surface protein B’). GspB export is mediated by a seven‐component accessory Sec system, containing two homologues of the general secretory pathway (SecA2 and SecY2) and five accessory Sec proteins (Asps1–5). Here we show that the Asps are required for optimal export of GspB independent of the glycosylation process. Furthermore, yeast two‐hybrid screening of the accessory Sec system revealed interactions occurring between Asp3 and the other components of the system. Asp3 was shown to bind SecA2, Asp1, Asp2 and itself. Mutagenesis of Asp3 identified N‐ and C‐terminal regions that are essential for GspB transport, and conserved residues within the C‐terminal domain mediated Asp3 binding to other accessory Sec components. The loss of binding by Asp3 also resulted in an impaired ability of S. gordonii to secrete GspB. These studies indicate that Asp3 is a central element mediating multiple interactions among accessory Sec components that are essential for GspB transport to the cell surface.     

Glycine residues in the hydrophobic core of the GspB signal sequence route export toward the accessory Sec pathway

The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.     

Two additional components of the accessory sec system mediating export of the Streptococcus gordonii platelet-binding protein GspB

The gspB-secY2A2 locus of Streptococcus gordonii strain M99 encodes the platelet-binding glycoprotein GspB, along with proteins that mediate its glycosylation and export. We have identified two additional components of the accessory Sec system (Asp4 and Asp5) encoded just downstream of gtfB in the gspB-secY2A2 locus. These proteins are required for GspB export and for normal levels of platelet binding by M99. Asp4 and Asp5 may be functional homologues of SecE and SecG, respectively.     

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.     

Sec2 is a highly efficient exchange factor for the Rab protein Sec4

Sec2 is a reversibly membrane associated multi-domain protein with guanine nucleotide exchange activity towards the yeast Rab-protein Sec4. Both proteins are localized to secretory vesicles destined for exocytosis. We have used transient kinetic methods to show that Sec2 is a highly active exchange factor, in contrast to other proteins previously characterized as Rab exchange factors. With a K(d) value for the Sec2:Sec4.GDP interaction of ca 70 microM and a maximal rate of GDP displacement of ca 15 s(-1), it is 100-1000-fold more effective than other proteins showing exchange activity towards Rabs (MSS4, DSS4, Vps9) and ca tenfold faster than Cdc25 as a Ras specific exchanger, although still 100-fold slower than the fastest systems studied so far, EF-Tu/Ef-Ts and Ran/RCC1. A comparison with other proteins showing Rab exchange activity shows that maximal rates of GDP dissociation catalyzed by Sec2 are orders of magnitude faster. When comparing Sec2 with DSS4, which also acts on Sec4, the difference was particularly dramatic. Another difference is seen in the kinetics of association of GTP with the Sec4:Sec2 complex, a process which is extremely slow for DSS4/MSS4 complexes with cognate Rabs but in the range observed for other GTPase:exchanger complexes for Sec4:Sec2., It is suggested that systems such as Ef-Tu/Ef-Ts and Ran/RCC1 have evolved for maximal possible activity for the interaction between two soluble proteins, whereas other evolutionary constraints which are connected to the spatial and temporal coordination of events in vesicular transport and other regulatory networks have determined the detailed kinetic properties of the other systems.     

Genes in the accessory sec locus of Streptococcus gordonii have three functionally distinct effects on the expression of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is strongly correlated with the expression of the large surface glycoprotein GspB. A 14 kb chromosomal region downstream of gspB was previously shown to be required for the expression of this protein. The region encodes SecA2 and SecY2, which are components of an accessory secretion system dedicated specifically to the export of GspB. The region also includes three genes (gly, nss and gtf) that encode proteins likely to function in carbohydrate metabolism, and four genes (orf1-4) that encode proteins of unknown function. In this report, we have investigated the role of these genes in GspB expression. We found that disruption of orf1, orf2 or orf3 resulted in a loss of GspB export and the intracellular accumulation of GspB. As they are apparently essential components of the accessory secretion system, these genes were renamed asp1-3 (for accessory secretory protein). In gtf and orf4 mutants, gspB was transcribed, but no GspB was detected. These results suggest that Gtf and Orf4 are required for the translation or for the stability of GspB. In contrast, gly and nss mutants were able to express and export GspB. However, disruption of these genes appeared to affect the carbohydrate composition of this glycoprotein. As asp1-3, gtf and orf4, but not gly and nss, are conserved in the accessory sec loci of several staphylococcal and streptococcal species, these genes may also have crucial roles in the expression and export of GspB homologues in the other Gram-positive bacteria.     

The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.     

The uncoupling protein 2 modulates the cytokine balance in innate immunity

The uncoupling protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.     

Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.     

The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast

SEC15 function is required at a late stage of the yeast secretory pathway. Duplication of the gene encoding the ras-like, GTP-binding protein, Sec4, can suppress the partial loss of function resulting from the sec15-l mutation, but cannot suppress disruption of sec15. Analysis of the SEC15 gene predicts a hydrophilic protein product of 105 kD. Anti-Sec15 antibody recognizes a protein of 116-kD apparent molecular mass which is associated with a microsomal fraction of yeast in a strongly pH dependent fashion. Overproduction of Sec15 protein interferes with the secretory pathway, resulting in the formation of a cluster of secretory vesicles, and a patch of Sec15 protein revealed by immunofluorescence. The sec4-8 and sec2-4l mutations, but not mutations in other SEC genes, prevent formation of the Sec15 protein patch. We propose that Sec15 protein responds to the function of the Sec4 protein to control vesicular traffic.     

An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets

The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are not required for viability or for the translocation of most exported proteins. The genes (secA2 and secY2) reside in a region of the chromosome required for the export of GspB, a 286 kDa cell wall-anchored protein. Loss of GspB surface expression is associated with a significant reduction in the binding of M99 to human platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99 has a second, canonical SecA homologue that is essential for viability. At least two other Gram-positive species, Streptococcus pneumoniae and Staphylococcus aureus, encode two sets of SecA and SecY homologues. One set is more similar to SecA and SecY of Escherichia coli, whereas the other set is more similar to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2-secA2 loci suggests that, in each of these Gram-positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine-rich repeat protein.     

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.     

The structure of the Sec complex and the problem of protein translocation

Proteins synthesized in the cytosol either remain there or are localized to a specific membrane and subsequently translocated to another cellular compartment. These extracytosolic proteins have to cross, or be inserted into, a phospholipid bilayer-a process governed by membrane-bound protein transporters designed to recognize and receive appropriate polypeptides and thread them through the membrane. One such translocation complex, SecY/Sec61, is found in every cell, in either the plasma membrane of bacteria and archaea or the endoplasmic reticulum membrane of eukaryotes. Recent structural findings, combined with previous genetic and biochemical studies, have helped to describe how the passage of proteins through the membrane might occur, but several points of uncertainty remain.     

The L7Ae RNA binding motif is a multifunctional domain required for the ribosome-dependent Sec incorporation activity of Sec insertion sequence binding protein 2

The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA([Ser]Sec) and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.     

The measles virus fusion protein transmembrane region modulates availability of an active glycoprotein complex and fusion efficiency

The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F₀) than activated (F₁₊₂) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-F^L507A, the F₀/F₁₊₂ ratio shifted toward F₁₊₂. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.     

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL^?1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.