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Biomedical subjects

Peter D Sun

Publications and source records attributed to Peter D Sun.

At least 19 recordsLinked to original sources
PubMed2006

An improved recombinant mammalian cell expression system for human transforming growth factor-beta2 and -beta3 preparations.

Transforming growth factor-beta2 and -beta3 (TGF-beta2 and -beta3) are important members of TGF-beta family which play important roles in the growth, maintenance, and repair processes of developing embryos, neonates, and adults. Preparation of large quantities of these two cytokines, which is necessary for structural studies and other applications, has proven to be extremely difficult. We have developed a novel Chinese hamster ovary cell-based expression system for high-level expression and high recovery of recombinant human TGF-beta2 and -beta3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-beta2 or -beta3 open reading frame for expression. The leader peptide of TGF-beta2 or -beta3 was replaced by that from the V-J2-C region of a mouse immunoglobulin kappa-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-beta2 or -beta3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-beta2 or -beta3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10 mg of TGF-beta2 and 8 mg of TGF-beta3 per liter of spent medium. The purification scheme involved the use of two simple chromatographic steps with a typical yield of 5 mg of TGF-beta2 and 4 mg of TGF-beta3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-beta superfamily members. We further confirmed that latent TGF-beta2 and -beta3 can be activated by proteolysis and glycolysis, which have not been reported before.

Animals↗
PubMed2006

A survey of protein-protein complex crystallizations.

A survey of crystallization conditions was carried out for 650 published protein-protein complexes in the Protein Data Bank (PDB) of the Research Collaboratory for Structural Bioinformatics (RCSB). This resulted in the establishment of a Protein Complex Crystallization Database (PCCD) and a set of configuration-space boundaries for protein-complex crystallizations. Overall, polyethylene glycol (PEG) based conditions accounted for 70-80% of all crystallizations, with PEG 3000-4000, 5000-6000 and 8000 being the most frequently used. The median values of PEG concentrations were between 10 and 20% and were inversely correlated with their molecular weights. Ammonium sulfate remained the most favorable salt precipitant, with a median concentration of 1.6 M. The crystallization pH for the vast majority of protein complexes was between 5.0 and 8.0. Overall, the boundaries for the crystallization configuration space of protein complexes appear to be more restricted than those of soluble proteins. This may reflect the limited stability and solubility of protein-protein complexes. Based on statistical analysis of the database, a sparse-matrix and a systematic buffer and pH screen were formulated to best represent the crystallization of protein complexes.

Buffers↗
PubMed2006

Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG.

Group A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.

Amino Acid Sequence↗
PubMed2005

Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors.

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein-Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide-MHC class I complexes on Epstein-Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders.

ATP-Binding Cassette Transporters↗
PubMed2005

The structure of DC-SIGNR with a portion of its repeat domain lends insights to modeling of the receptor tetramer.

The dendritic cell-specific ICAM-3 non-integrin (DC-SIGN) and its close relative DC-SIGNR recognize various glycoproteins, both pathogenic and cellular, through the receptor lectin domain-mediated carbohydrate recognition. While the carbohydrate-recognition domains (CRD) exist as monomers and bind individual carbohydrates with low affinity and are permissive in nature, the full-length receptors form tetramers through their repeat domain and recognize specific ligands with high affinity. To understand the tetramer-based ligand binding avidity, we determined the crystal structure of DC-SIGNR with its last repeat region. Compared to the carbohydrate-bound CRD structure, the structure revealed conformational changes in the calcium and carbohydrate coordination loops of CRD, an additional disulfide bond between the N and the C termini of the CRD, and a helical conformation for the last repeat. On the basis of the current crystal structure and other published structures with sequence homology to the repeat domain, we generated a tetramer model for DC-SIGN/R using homology modeling and propose a ligand-recognition index to identify potential receptor ligands.

Amino Acid Sequence↗
PubMed2005

Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy.

Natural killer (NK) cells play an important role in both innate and adaptive antiviral immune responses. The adaptive response typically requires that virus-specific antibodies decorate infected cells which then direct NK cell lysis through a CD16 mediated process termed antibody-dependent cellular cytotoxicity (ADCC). In this report, we employ a highly polymerized chimeric IgG1/IgA immunoglobulin (Ig) fusion protein that, by virtue of its capacity to extensively crosslink CD16, activates NK cells while directing the lysis of infected target cells. We employ HIV as a model system, and demonstrate that freshly isolated NK cells preloaded with an HIV gp120-specific chimeric IgG1/IgA fusion protein efficiently lyse HIV-infected target cells at picomolar concentrations. NK cells pre-armed in this manner retain the capacity to kill targets over an extended period of time. This strategy may have application to other disease states including various viral infections and cancers.

Antibody-Dependent Cell Cytotoxicity↗
PubMed2005

Characterization of DC-SIGN/R interaction with human immunodeficiency virus type 1 gp120 and ICAM molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor.

The dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin binding receptor (DC-SIGN) was shown to bind human immunodeficiency virus type 1 (HIV-1) viral envelope protein gp120 and proposed to function as a Trojan horse to enhance trans-virus infection to host T cells. To better understand the mechanism by which DC-SIGN and DC-SIGNR selectively bind HIV-1 gp120, we constructed a series of deletion mutations in the repeat regions of both receptors. Different truncated receptors exist in different oligomeric forms. The carbohydrate binding domain without any repeats was monomeric, whereas the full extracellular receptors existed as tetramers. All reconstituted receptors retained their ability to bind gp120. The dissociation constant, however, differed drastically from micromolar values for the monomeric receptors to nanomolar values for the tetrameric receptors, suggesting that the repeat region of these receptors contributes to the avidity of gp120 binding. Such oligomerization may provide a mechanism for the receptor to selectively recognize pathogens containing multiple high-mannose-concentration carbohydrates. In contrast, the receptors bound to ICAMs with submicromolar affinities that are similar to those of two nonspecific cell surface glycoproteins, FcgammaRIIb and FcgammaRIII, and the oligomerization of DC-SIGNR resulted in no increase in binding affinity to ICAM-3. These findings suggest that DC-SIGN may not discriminate other cell surface glycoproteins from ICAM-3 binding. The pH dependence in DC-SIGN binding to gp120 showed that the receptor retained high-affinity gp120 binding at neutral pH but lost gp120 binding at pH 5, suggesting a release mechanism of HIV in the acidic endosomal compartment by DC-SIGN. Our work contradicts the function of DC-SIGN as a Trojan horse to facilitate HIV-1 infection; rather, it supports the function of DC-SIGN/R (a designation referring to both DC-SIGN and DC-SIGNR) as an antigen-capturing receptor.

Animals↗
PubMed2004

Insight of host immune evasion mediated by two variants of group a Streptococcus Mac protein.

Group A Streptococcus has evolved numerous mechanisms to evade the host immune system to survive, disseminate, and cause disease. Recently a secreted protein named Mac-1 was identified and shown to enhance survival of the pathogen. A new variant of Mac-1 (designated Mac-2) also was recently described and shown to differ from Mac-1 by approximately 50% amino acid sequence divergence in the middle one-third of the molecule. To gain new information about the role of Mac-1 and Mac-2 in host-pathogen interactions, solution binding experiments were performed using surface plasmon resonance and purified Mac proteins. Mac-1 bound the same lower hinge region of human IgG as Fc receptors with 2.5 microM affinity, which lead to proteolytic cleavage of the antibody. Similar Km (6.8-18.9 microM) and kcat (0.02-0.13 s(-1)) values of the Mac-1 endopeptidase activity were obtained for IgG1, IgG2, IgG3, and IgG4. Mac-2 variant, in contrast, bound human IgG poorly (KD = 16 mM) and had weak endopeptidase activity against IgG. Instead, Mac-2 bound FcgammaRII and FcgammaRIII with 5 and 75 microM affinity, respectively. This binding competitively blocked IgG from recognition by Fc receptors. Taken together, Mac proteins block immunoglobulin recognition by Fc receptors and degrade immunoglobulins, thereby enhancing survival of the pathogen through the inhibition of phagocytosis, endocytosis of IgG-opsonized particles, and antibody-dependent cell-mediated cytotoxicity. Consequently, these proteins may be potential therapeutic targets.

Amino Acid Sequence↗
PubMed2004

Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system.

Transforming growth factor-beta1 (TGF-beta1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-beta1 disulfide bonded to latent TGF-beta1-binding protein (LTBP). Current recombinant expression systems yield less than 1-2 mg of the mature TGF-beta1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-beta1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-beta1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-beta1 per liter of spent medium. Purified TGF-beta1 bound with high affinity to its type II receptor with a solution dissociation constant of approximately 70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-beta family members, this recombinant expression system may also be applied to the overexpression of other TGF-beta isomers and even to members of the TGF-beta superfamily to facilitate their preparation.

Animals↗
PubMed2003

Crystal structure of the human natural killer (NK) cell activating receptor NKp46 reveals structural relationship to other leukocyte receptor complex immunoreceptors.

Natural cytotoxicity receptors (NCR) mediate lysis of a variety of tumor and virus-infected cells by natural killer (NK) cells. Upon engagement, NCR trigger the cytolytic activity and cytokine release of NK cells through association with ITAM-containing signaling molecules. To further understand the function of these receptors in activation of natural cytotoxicity, we determined the crystal structure of the extracellular ligand binding domain of human NKp46, one of three known NCR, at 2.2-A resolution. The overall fold and disposition of the two C2-set immunoglobulin domains are similar to the D1D2 domains of inhibitory killer cell Ig-like receptor (KIR) and Ig-like transcript (ILT) receptors. As the cellular ligands of NKp46 have not yet been defined, the known ligand binding sites of KIR and ILT were compared with the corresponding structural regions of NKp46 in an effort to identify potential areas suitable for molecular recognition. A potential binding site for influenza hemagglutinin is located near the interdomain hinge, a region that mediates ligand binding in KIR. The structural similarity of NKp46 to inhibitory KIR receptors may have implications for how NK cells balance activating and inhibitory signals.

Amino Acid Sequence↗
PubMed2003

Crystal structure of the human myeloid cell activating receptor TREM-1.

Triggering receptors expressed on myeloid cells (TREM) are a family of recently discovered receptors that play important roles in innate immune responses, such as to activate inflammatory responses and to contribute to septic shock in response to microbial-mediated infections. To date, two TREM receptors in human and several homologs in mice have been identified. We report the 2.6 A resolution crystal structure of the extracellular domain of human TREM-1. The overall fold of the receptor resembles that of a V-type immunoglobulin domain with differences primarily located in the N-terminal strand. TREM-1 forms a "head-to-tail" dimer with 4100 A(2) interface area that is partially mediated by a domain swapping between the first strands. This mode of dimer formation is different from the "head-to-head" dimerization that existed in V(H)V(L) domains of antibodies or V domains of T cell receptors. As a result, the dimeric TREM-1 most likely contains two distinct ligand binding sites.

Amino Acid Sequence↗
PubMed2003

Structure and function of natural-killer-cell receptors.

The function of natural-killer (NK) cells is modulated by the balance between a number of activating and inhibitory receptors. Killer immunoglobulinlike receptors (KIRs) are mostly inhibitory receptors. They play a critical role in recognizing self-class-I major histocompatibility complex (MHC) molecules and thus protect healthy host cells from NK-targeted lysis. In contrast, both NKG2D and CD16 are activating NK receptors that trigger the NK-cell lysis of various tumor and virally infected cells through either direct ligand engagement or antibody-dependent cellular cytotoxicity (ADCC). Through structural studies of members of these distinct receptor families, in particular, the structure and recognition between KIR2DL2 and HLA-Cw3, that between NKG2D and ULBP3, and that between CD16 and IgG Fc, considerable understandings have been achieved about their function and their ligand recognition.

Animals↗
PubMed2002

Structure and function of natural killer cell surface receptors.

Since mid-1990, with cloning and identification of several families of natural killer (NK) receptors, research on NK cells began to receive appreciable attention. Determination of structures of NK cell surface receptors and their ligand complexes led to a fast growth in our understanding of the activation and ligand recognition by these receptors as well as their function in innate immunity. Functionally, NK cell surface receptors are divided into two groups, the inhibitory and the activating receptors. Structurally, they belong to either the immunoglobulin (Ig)-like receptor superfamily or the C-type lectin-like receptor (CTLR) superfamily. Their ligands are either members of class I major histocompatibility complexes (MHC) or homologs of class I MHC molecules. The inhibitory form of NK receptors provides the protective immunity through recognizing class I MHC molecules with self-peptides on healthy host cells. The activating, or the noninhibitory, NK receptors mediate the killing of tumor or virally infected cells through their specific ligand recognition. The structures of activating and inhibitory NK cell surface receptors and their complexes with the ligands determined to date, including killer immunoglobulin-like receptors (KIRs) and their complexes with HLA molecules, CD94, Ly49A, and its complex with H-2Dd, and NKG2D receptors and their complexes with class I MHC homologs, are reviewed here.

Antigen-Antibody Complex↗
PubMed2002

Making sense of the diverse ligand recognition by NKG2D.

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.

Animals↗
PubMed2002

Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases.

Screening for heavy-atom derivatives remains a time-consuming and cumbersome process that often results in non-isomorphous derivatives whose phases cannot be combined. Using lysozyme and FcgammaRIII receptor crystals as test cases, an improved soaking method for the generation of conventional heavy-atom derivatives has been developed. The method is based on soaking crystals in heavy-atom compounds for a very brief time at near-saturation concentrations. Compared with the current heavy-atom soaking method, which often takes days to achieve a derivatization, the quick-soak method completes a derivatization within 10 min to 2 h. The bound heavy-atom sites display higher peak heights from quick soaks than from overnight soaks in all cases tested. The quick-soak derivatives also preserved native-like diffraction resolution and data quality that was better than the prolonged-soak derivatives. Furthermore, derivatives generated by brief soaks are more isomorphous to the native than those generated by overnight soaks. Short soaks not only increase the likelihood of success in heavy-atom screening by reducing the pitfalls associated with prolonged soaks, such as lack of isomorphism and overall lattice disorder, but also have the potential to transform a time-consuming derivative screening into an 'on-the-fly' real-time derivatization process.

Animals↗
PubMed2002

Generating isomorphous heavy-atom derivatives by a quick-soak method. Part II: phasing of new structures.

A quick-soak method has been applied to generate de novo heavy-atom phasing to solve two new protein structures, a type II transforming growth factor beta receptor (TBRII) and a natural killer cell receptor-ligand complex, NKG2D-ULBP3. In the case of TBRII, a crystal derivatized for only 10 min in saturated HgCl(2) provided adequate phasing for structure determination. Comparison between HgCl(2) derivatives generated by 10 min soaking and by 12 h soaking revealed similar phasing statistics. The shorter soak, however, resulted in a derivative more isomorphous to the native than the longer soak as judged by changes in the unit-cell parameter a upon derivatization as well as by the quality of a combined SIRAS electron-density map. In the case of the NKG2D-ULBP3 structure, all overnight soaks in heavy-atom solutions resulted in crystal lattice disorder and only the quick soaks preserved diffraction. Despite fragile lattice packing, the quick-soaked K(2)PtCl(4) derivative was isomorphous with the native crystal and the electron-density map calculated from combined SIR and MAD phases is better than that calculated from MAD phases alone. Combined with mass-spectrometry-assisted solution heavy-atom derivative screening and the use of synchrotron radiation, the quick-soak derivatization has the potential to transform the time-consuming conventional heavy-atom search into a real-time 'on-the-fly' derivatization process that will benefit high-throughput structural genomics.

Carrier Proteins↗