A Single Residue Determines the Cooperative Binding Property of a Primosomal DNA Replication Protein,PriB, to Single-Stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)₂₅, and (StPriB)₂₅ are proposed.     

Involvement of histidine in complex formation of PriB and single-stranded DNA

PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, ¹H or ¹⁵N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB–ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8 nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57–Ser65) and in β4–loop3–β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5′-Cy3- and 3′-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB–ssDNA complex and on the positive cooperativity of PriB.     

Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 Å resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB–dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB–ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L₄₅ plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA–PriB–ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.     

Purine nucleoside phosphorylase from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. Bsi590: molecular cloning,gene expression and characterization of the recombinant protein

The gene encoding purine nucleoside phosphorylase (PNP) from the cold-adapted marine bacterium Pseudoalteromonas sp. Bsi590 was identified, cloned and expressed in Escherichia coli. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. Pseudoalteromonas sp. Bsi590 PNP (PiPNP) shares 60% amino sequence identity and conservation of amino acid residues involved in catalysis with mesophilic Escherichia coli deoD-encoded purine nucleoside phosphorylase (EcPNP). N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni²⁺-chelating column. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties; PiPNP possessed higher K _m and catalytic efficiency (k _cat/K _m ) compared to EcPNP at 37°C. Substrate specificity results showed PiPNP catalyzed the phosphorolytic cleavage of 6-oxopurine and 6-aminopurine nucleosides (or 2-deoxynucleosides), and to a lesser extent purine arabinosides. PiPNP showed a better activity with inosine while no activity toward pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum. Results showed that PiPNP had lower conformation transition point temperature than EcPNP; phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability.     

The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.     

A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11-389

The protection of telomeres 1 (Pot1) proteins specifically recognize the single-stranded 3' end of the telomere, an activity essential for sustained cellular viability and proliferation. The current model for the telomeric single-stranded DNA (ssDNA) binding activity of Schizosaccharomyces pombe Pot1 is based on a 20 kDa fragment, Pot1pN. Recent biochemical studies suggest that SpPot1 contains a larger ssDNA-binding domain and we have identified a novel ssDNA-binding domain similar in size to the human Pot1 domain. This domain, Pot1(1-389), binds extremely tightly to an oligonucleotide consisting of two conserved hexameric S. pombe telomere repeats, d(GGTTACGGTTAC), with an affinity approximately 4000-fold tighter than Pot1pN binds its cognate ssDNA. The Pot1(1-389)/ssDNA complex exhibits a half-life of 53 min, consistent with that estimated for full-length SpPot1 and significantly longer than that of Pot1pN. Single nucleotide substitutions reveal that, in contrast to Pot1pN, tandem trinucleotide repeats (GTT) within d(GGTTACGGTTAC) are specifically recognized by Pot1(1-389). Interestingly, certain single nucleotide substitutions that impacted Pot1pN binding exhibited no effect on binding affinity by Pot1(1-389). However, these substitutions reduced binding affinity when simultaneously substituted in each hexameric repeat. The non-additive nature of these substitutions suggests that certain nucleotides are coupled through the ability of the flexible ssDNA oligonucleotide to adopt alternate, thermodynamically equivalent conformations. The biochemical behavior of Pot1(1-389) is more similar to that of the full-length SpPot1 protein than to that of Pot1pN, making Pot1(1-389) a valuable domain for the future study of how full-length SpPot1 interacts with telomeric ssDNA.     

Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells

The replacement of Escherichia coli recA gene (recA_Ec) with the Pseudomonas aeruginosa recA_Pa gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA_Pa protein with those of RecA_Ec protein. Consistent with hyper-recombination activity, RecA_Pa protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA_Pa protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA_Ec protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA_Pa protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA_Pa in comparison with RecA_Ec, RecA441 and RecA803 proteins, RecA_Pa showed unusually high abilities: to be resistant to the displacement by SSB protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA_Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.     

A single residue determines the cooperative binding property of a primosomal DNA replication protein, PriB, to single-stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed.     

Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer

Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt²/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.     

Cys-92, Cys-95, and the C-terminal 12 residues of the <Emphasis Type="Italic">Vibrio harveyi</Emphasis> ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur_vh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur_Ec) and could complement a mutant of Fur_Ec. Like Fur_Ec, Fur_Vh, possesses two cysteine residues at positions 92 and 95, yet unlike Fur_Ec, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur_Vh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur_vh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur_Vh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur_Vh is possibly different from that of Fur_Ec; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur_Vh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k_cat regulation. Surprisingly, the T11A mutant had a decreased GSH K_m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.     

Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.     

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.

Summary The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.Abbreviations RecAwt RecA wind-type protein - ssDNA singlestranded DNA - dsDNA dmble-stranded DNA     

Insight into the interaction between PriB and DnaT on bacterial DNA replication restart: Significance of the residues on PriB dimer interface and highly acidic region on DnaT

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB−ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66−Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria.     

High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner

The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration–HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1–3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC₅₀s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.