Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.
Small disulfide-bonded proteins (SDPs) are rich sources for therapeutic drugs. Designing drugs from these proteins requires three-dimensional structural information, which is only available for a subset of these proteins. SDPMOD addresses this deficit in structural information by providing a freely available automated comparative modeling service to the research community. For expert users, SDPMOD offers a manual mode that permits the selection of a desired template as well as a semi-automated mode that allows users to select the template from a suggested list. Besides the selection of templates, expert users can edit the target–template alignment, thus allowing further customization of the modeling process. Furthermore, the web service provides model stereochemical quality evaluation using PROCHECK. SDPMOD is freely accessible to academic users via the web interface at http://proline.bic.nus.edu.sg/sdpmod.
Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.
The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments 1 . Among amniotes, genome sequences are available for mammals and birds 2–4 , but not for non-avian
The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of ‘domain’ do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.
Sequences at the 3'-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3'-terminal region of negative RNA strand ((-)3'T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3'-cytidylate (3'C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3'-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3'C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.
We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.
The leukocyte β2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of β2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the β2 inserted (I)-like domain. This mutation converted αLβ2 from a resting into a high affinity conformer because αLβ2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by αLβ2N329S because of its reactivity with the β2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation inβ3 generated constitutively activeαIIbβ3 that adhered to fibrinogen. This Asn is conserved in all human β subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of theα7 helix and the β6-α7 loop in the I-like domain. The leukocyte β2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of β2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the β2 inserted (I)-like domain. This mutation converted αLβ2 from a resting into a high affinity conformer because αLβ2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by αLβ2N329S because of its reactivity with the β2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation inβ3 generated constitutively activeαIIbβ3 that adhered to fibrinogen. This Asn is conserved in all human β subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of theα7 helix and the β6-α7 loop in the I-like domain. The integrins are type I membrane cell adhesion molecules formed non-covalently by two subunits (α/β). Despite having no intrinsic enzymatic properties, integrins are bidirectional signal transducers brought about by the recruitment of cytosolic proteins, many of which are signaling proteins, to their relatively short cytoplasmic tails (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6884) Google Scholar). Structural data reveal a composite of distinct domains and folds found in an integrin molecule (2Arnaout M.A. Mahalingam B. Xiong J.P. Annu. Rev. Cell Dev. Biol. 2005; 21: 381-410Crossref PubMed Scopus (419) Google Scholar, 3Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1247) Google Scholar). Out of the 24 human integrins, nine contain in the α subunit an additional inserted (I) domain that is the primary ligand-binding domain of these integrins (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6884) Google Scholar). The I domain has a metal ion-dependent adhesion site (MIDAS) 3The abbreviations used are: MIDAS, metal ion-dependent adhesion site; ADMIDAS, adjacent to MIDAS; LIMBS, ligand-induced metal-binding site; ICAM, intercellular adhesion molecule; LAD-1, leukocyte adhesion deficiency-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline; I, inserted; EI, expression index; PDB, Protein Data Bank. that contains a divalent cation essential for ligand binding. Integrins that lack the I domain (henceforth referred to as I-less integrins) are found to bind ligand via the β propeller of their α subunit and the I-like domain of their β subunit (2Arnaout M.A. Mahalingam B. Xiong J.P. Annu. Rev. Cell Dev. Biol. 2005; 21: 381-410Crossref PubMed Scopus (419) Google Scholar, 3Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1247) Google Scholar). The I-like domain is found in all integrin β subunits, and it is structurally similar to that of the I domain. However, it contains a specificity-determining loop, which was reported to contribute toward ligand binding specificity and integrin αβ subunit association (4Takagi J. DeBottis D.P. Erickson H.P. Springer T.A. Biochemistry. 2002; 41: 4339-4347Crossref PubMed Scopus (59) Google Scholar), and it has two additional divalent cation-binding sites. The MIDAS of the I-like domain is flanked by the adjacent to MIDAS (ADMIDAS) and the ligand-induced metal-binding site (LIMBS), which serve as negative and positive regulatory sites, respectively (5Chen J. Salas A. Springer T.A. Nat. Struct. Biol. 2003; 10: 995-1001Crossref PubMed Scopus (123) Google Scholar, 6Chen J. Takagi J. Xie C. Xiao T. Luo B.H. Springer T.A. J. Biol. Chem. 2004; 279: 55556-55561Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 7Mould A.P. Barton S.J. Askari J.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 51622-51629Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The conserved coordinating residues that are involved in the three cation-binding sites of the I-like domain are highlighted (Fig. 1A). The leukocyte-restricted β2 integrins contain four members that differ in their α subunits, the αLβ2, αMβ2, αXβ2, and αDβ2 (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6884) Google Scholar). These integrins maintain a functional immune system by their direct involvement in processes such as leukocyte adhesion and migration, phagocytosis, and antigen presentation. All four members contain the I domain that serves as the primary ligand-binding site. The structure of an I domain-containing integrin is lacking. Thus, the mechanism of I domain regulation by other domain(s) in an intact integrin is drawn largely from isolated I domain structures without or with ligands (8Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 9Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (804) Google Scholar, 10Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 11Shimaoka M. Xiao T. Liu J.H. Yang Y. Dong Y. Jun C.D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), cogent mutagenesis studies that sculpted different I domain conformers reporting different ligand binding affinities (12Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (189) Google Scholar, 13Shimaoka M. Lu C. Salas A. Xiao T. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16737-16741Crossref PubMed Scopus (57) Google Scholar), and extrapolation of possible I domain connectivity with other domain(s) from known structures of I-less integrins (14Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1113) Google Scholar, 15Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (682) Google Scholar). Notably, I domain ligand binding was shown to be regulated allosterically by the I-like domain. An invariant glutamate residing in the last helix of the I domain serves as an intrinsic ligand for the I-like domain (16Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (78) Google Scholar). Mutations in the I-like domain of the β2 integrins are known to generate receptors with impaired functions as exemplified in the inherited disorder leukocyte adhesion deficiency 1 (LAD-1) (17Hogg N. Bates P.A. Matrix Biol. 2000; 19: 211-222Crossref PubMed Scopus (82) Google Scholar). The I-like domain mutations S116P and D209H generate β2 integrins that were expressed on the cell surface but dysfunctional (18Hogg N. Stewart M.P. Scarth S.L. Newton R. Shaw J.M. Law S.K. Klein N. J. Clin. Investig. 1999; 103: 97-106Crossref PubMed Scopus (145) Google Scholar, 19Mathew E.C. Shaw J.M. Bonilla F.A. Law S.K. Wright D.A. Clin. Exp. Immunol. 2000; 121: 133-138Crossref PubMed Scopus (47) Google Scholar). Ser116 and Asp209 are coordinating residues of the MIDAS/ADMIDAS and LIMBS, respectively (Fig. 1A). Interestingly, the I-like domain mutation N329S, which was not inherited from either parent, was identified in a patient having another mutation causing aberrant splicing of the integrin β2 subunit (20Nelson C. Rabb H. Arnaout M.A. J. Biol. Chem. 1992; 267: 3351-3357Abstract Full Text PDF PubMed Google Scholar). Although N329S mutation supported moderate integrin αMβ2 expression in a surrogate cell transfection system (20Nelson C. Rabb H. Arnaout M.A. J. Biol. Chem. 1992; 267: 3351-3357Abstract Full Text PDF PubMed Google Scholar), it remains unclear as to the effect of this mutation on β2 integrin ligand binding function. Previously, we reported the expression of a constitutively active αLβ2N329S that adhered to intercellular adhesion molecule (ICAM)-1 (21Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Accumulating evidence suggests that integrin αLβ2 may undergo conformational changes that generate the resting, intermediate, and high affinity states (22Tang R.H. Tng E. Law S.K. Tan S.M. J. Biol. Chem. 2005; 280: 29208-29216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Nishida N. Xie C. Shimaoka M. Cheng Y. Walz T. Springer T.A. Immunity. 2006; 25: 583-594Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Herein, we further characterize and report N329S as a mutation that promotes a high affinity αLβ2. Similarly, the same mutation introduced into the I-less integrin αIIbβ3 produced an active receptor. Combinatorial analyses of N329S with S116P or D209H showed that the activating effect of N329S requires functional I-like domain MIDAS and LIMBS to allow propagation of the activating signal to the αL I domain. Of note, this Asn at position 329 in integrin β2 primary sequence is conserved in all integrin β subunits. It may interact with neighboring residues to stabilize the β I-like domain β6-α7 loop that is required for the transmission of activation signal. Reagents and mAbs—Recombinant human ICAM-1/Fc and ICAM-3/Fc were prepared as described previously (24Simmons D.L. Hartley D. Cellular Interactions in Development. Oxford University Press, UK1993: 93-127Google Scholar). Human fibrinogen was obtained from Sigma. These mAbs were kind gifts from different sources: MHM24 (αLβ2-specific and function-blocking) (25Hildreth J.E. Gotch F.M. Hildreth P.D. McMichael A.J. Eur. J. Immunol. 1983; 13: 202-208Crossref PubMed Scopus (234) Google Scholar) and MHM23 (β2 integrin heterodimer-specific) (26Hildreth J.E. August J.T. J. Immunol. 1985; 134: 3272-3280PubMed Google Scholar) were from A. J. McMichael (Institute of Molecular Medicine, Oxford, UK); KIM185 (β2 integrin-activating mAb) (27Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar) and KIM127 (β2 integrin-specific and activation reporter mAb) (28Stephens P. Romer J.T. Spitali M. Shock A. Ortlepp S. Figdor C.G. Robinson M.K. Cell Adhes. Commun. 1995; 3: 375-384Crossref PubMed Scopus (48) Google Scholar) were from M. K. Robinson (CellTech, Slough, UK); and 10E5 (integrin αIIb-specific and function-blocking) (29Coller B.S. Peerschke E.I. Scudder L.E. Sullivan C.A. J. Clin. Investig. 1983; 72: 325-338Crossref PubMed Scopus (562) Google Scholar, 30Luo B.H. Takagi J. Springer T.A. J. Biol. Chem. 2004; 279: 10215-10221Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and 7E3 (β3 integrin recognizing mAb) (31Artoni A. Li J. Mitchell B. Ruan J. Takagi J. Springer T.A. French D.L. Coller B.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13114-13120Crossref PubMed Scopus (75) Google Scholar) were from B. S. Coller (The Rockefeller University, New York, NY). The mAb MEM148 (β2 integrin-specific and hybrid domain displacement reporter mAb) (22Tang R.H. Tng E. Law S.K. Tan S.M. J. Biol. Chem. 2005; 280: 29208-29216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) was purchased from Serotec, Oxford, UK. cDNAs, Expression Plasmids, and mAbs—The αL, αM, αX and β2 pcDNA3 expression plasmids were described previously (32Shaw J.M. Al-Shamkhani A. Boxer L.A. Buckley C.D. Dodds A.W. Klein N. Nolan S.M. Roberts I. Roos D. Scarth S.L. Simmons D.L. Tan S.M. Law S.K. Clin. Exp. Immunol. 2001; 126: 311-318Crossref PubMed Scopus (44) Google Scholar). The αIIb and β3 pcDNA3 expression plasmids were kindly provided by P. J. Newman, Blood Center of Wisconsin and Medical College Wisconsin. Amino acid numbering of the integrins is based on Barclay et al. (33Barclay A.N. Brown M.H. Law S.K. McKnight A.J. Tomlinson M.G. van der Merwe P.A. The Leucocyte Antigen Facts Book. Second Ed. Academic press, London, UK1997Google Scholar). All integrin mutants were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the relevant primer pair. Integrins with more than one mutation (e.g. β2N329S/D209H) were generated by sequential site-directed mutagenesis. The αLc-c construct was generated by mutating I domain Lys287 and Lys294 into Cys to allow disulfide bridge formation (12Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (189) Google Scholar). All constructs were verified by sequencing (Research Biolabs, Singapore). Cell Transfections and Expression Analyses—Human embryonic kidney 293T cells were obtained from ATCC (Manassas, VA), and maintained in growth medium Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10%(v/v) heat-inactivated fetal bovine serum (Sigma), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich) at 37 °C in a 5% CO2 humidified atmosphere. 293T cells were transfected with expression plasmids using the calcium phosphate method (34DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (915) Google Scholar). Flow Cytometry Analyses—The preparation of samples for flow cytometry analyses was reported previously (35Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (55) Google Scholar). Cell surface expression of β3 and β2 integrins was analyzed using the mAb 7E3 and the heterodimer-specific mAb MHM23, respectively, followed by flow cytometry analysis on a FACSCalibur using the software CellQuest (BD Biosciences). The expression level was represented by the expression index (EI) that was calculated by the percentage of cells gated positive × geo-mean fluorescence intensity. An irrelevant mAb was used as background control in all preparations. Cell Adhesion Assays—Adhesion of αLβ2 transfectants to ICAMs was performed as reported (35Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (55) Google Scholar). Briefly, each Polysorb microtiter well (Nunc, Rosklide, Denmark) was coated with 0.5 μg of goat anti-human IgG (Fc specific) (Sigma) in 100 μl of 50 mm bicarbonate buffer (pH 9.2). Nonspecific binding sites were blocked with 0.5% (w/v) bovine serum albumin in PBS for 30 min at 37 °C. Thereafter, 50 μl of ICAM-Fc at 1 ng/μl in PBS containing 0.1% (w/v) bovine serum albumin was added to each well and incubated for 2 h at room temperature. Wells were washed twice in RPMI wash buffer (RPMI medium containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mm HEPES, pH 7.4) before assay. Cells labeled with 3.0 mm 2′,7′ bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescent dye (Molecular Probes, Eugene, OR) were incubated in wash buffer containing Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) and/or activating mAb KIM185 (10 μg/ml) to activate αLβ2. αLβ2-mediated adhesion specificity was demonstrated using MHM24 (10 μg/ml). Fluorescence signal, which correlates with the number of cells adhering to the ligand-coated well, is measured using a FL600 fluorescent plate reader (Bio-Tek instruments, Winooski, VT). For αIIbβ3 transfectant adhesion to fibrinogen, fibrinogen at 1 μg/ml in PBS was added into each microtiter well. The subsequent steps in the binding assay were the same as that of αLβ2 aforementioned. αIIbβ3-mediated adhesion specificity was assessed using the function-blocking mAb 10E5 (10 μg/ml). 1 mm MnCl2 was used to activate αIIbβ3. Surface Labeling and Immunoprecipitation—Surface labeling of integrins with biotin was described previously (35Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (55) Google Scholar). Cells were washed once in PBS and incubated in sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was terminated by washing surface-labeled cells once in PBS containing 10 mm Tris-HCl (pH 8.0). Thereafter, labeled cells were incubated in warm culture medium containing appropriate mAb MHM23, KIM127, or MEM148 (2 μg each) in the absence or presence of Mg/EGTA for 30 min at 37 °C. Cells were spun down and lysed in lysis buffer (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 1% (v/v) Nonidet P-40) containing appropriate protease inhibitors (Roche Diagnostics, Basel, Switzerland) followed by immunoprecipitation with rabbit anti-mouse IgG coupled to Protein A-Sepharose beads (Amersham Biosciences, Buckinghamshire, UK). Bound proteins were resolved on a 7.5% SDS-PAGE gel under reducing conditions. Proteins were transferred onto Immobilon P membrane (Millipore, Bedford, MA), and biotinylated protein bands were detected with streptavidin-horseradish peroxidase followed by enhanced chemiluminescence detection using the ECL Plus kit (Amersham Biosciences). Structural Images and Modeling—LSQKAB (Collaborative Computational Project, CCP4) (36Collaborative Computational Project Number 4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19764) Google Scholar) was used for molecular least-squares superposition of the three I-like domain conformers. Figs. 1B and 7 were created using PyMOL. The solvent-accessible surface area of β3 Asn339 of the three conformers was calculated using AREAIMOL (Collaborative Computational Project Number 4) with a probe of 1.7 Å radius: 46.1 Å2 (conformer I); 16.5 Å2 (conformer II); 68.8 Å2 (conformer III). Structural models of wild-type β2 I-like domain or variants were generated using Modeler 9.1. Energy computations of wild-type β2 I-like domain model or variants having Asn329 substituted with Gln, Ala, Ser, Thr, or Asp were done with GROMOS96 implementation of Swiss-PdbViewer. The models generated were examined for potential hydrogen bond(s) formation between Gln329, Ala329, Ser329, Thr329, or Asp329 of the α7 helix with Ser324 and Glu322 of the β6 strand. In the wild-type β2 I-like domain, Asn329 δ1O and δ2NH2 hydrogen-bond with Ser324 and main chain carbonyl of Glu322, respectively. In variant N329Q, Gln329ϵ1O hydrogen-bonds with Ser324. The variants N329A, N329S, and N329T lack a hydrogen bond between Asn329 and Ser324 or Glu322. In variant N329D, Asp329 hydrogen-bonds with Ser324 but not with Glu322. N329S Generates a High Affinity αLβ2 That Adheres to ICAM-1 and ICAM-3 Substrates Constitutively—The possibility of at least three affinity states of αLβ2 based on functional and structural studies prompted us to examine the affinity state of αLβ2N329S (22Tang R.H. Tng E. Law S.K. Tan S.M. J. Biol. Chem. 2005; 280: 29208-29216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Nishida N. Xie C. Shimaoka M. Cheng Y. Walz T. Springer T.A. Immunity. 2006; 25: 583-594Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). We showed previously that an intermediate affinity αLβ2 adhered to ICAM-1 but that a high affinity conformer was required for adhesion to ICAM-3 (22Tang R.H. Tng E. Law S.K. Tan S.M. J. Biol. Chem. 2005; 280: 29208-29216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Herein, αLβ2N329S transfectant showed a high level of constitutive adhesion to ICAM-1 even in the absence of activating agent, whereas wild-type αLβ2 required activation with Mg/EGTA (Fig. 2). This is consistent with our first report on the constitutive activity of αLβ2 N329S with respect to ICAM-1 adhesion (21Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Importantly, unlike wild-type αLβ2 transfectant, αLβ2N329S transfectant also showed a high level of constitutive adhesion to ICAM-3 in the absence of activating agents Mg/EGTA and the integrin β2-specific activating mAb KIM185 (Fig. 2). The expressions (represented as EI) of wild-type αLβ2 and αLβ2N329S were comparable as assessed by flow cytometry using the β2 integrin heterodimer-specific mAb MHM23. The binding specificity was demonstrated using the αLβ2-specific function-blocking mAb MHM24. Thus, the mutation N329S generates a high affinity αLβ2 with respect to ICAMs adhesions. The Requirement of Cβ Instead of Cγ Amide Functional Group at Position 329 of the β2 I-like Domain—The Asn at position 329 of the β2 is conserved in all integrin β subunits. Because the native structure of the β2 I-like domain has not been resolved and because of the fact that the corresponding residue in the β3 is Asn339, we made use of the resolved structure of the β3 I-like domain of αVβ3 in complex with an Arg-Gly-Asp (RGD) ligand (37Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1402) Google Scholar) to visualize the position of this conserved Asn and the three metal-binding sites. The β3 Asn339 lies before the last helix of the I-like domain (Fig. 1B). Further, the position of LIMBS (gold), MIDAS (pink), and ADMIDAS (blue) cations and the location of Asp217 (coordinating residue for LIMBS) and Ser123 (coordinating residue for MIDAS) are illustrated. In the liganded I domain, a significant downward displacement of its C-terminal helix was observed (8Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 38Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar). Consistent with this observation, open conformation αL and αM I domains, which have high affinity ligand binding properties, were generated by introducing cystine that stabilized the last helix of the I domains (12Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (189) Google Scholar, 13Shimaoka M. Lu C. Salas A. Xiao T. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16737-16741Crossref PubMed Scopus (57) Google Scholar). Similar to the I domain, the last helix of the β3 I-like domain was displaced in a ligand mimetic-bound αIIbβ3 (15Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (682) Google Scholar). At present, it is unclear how β2N329S confers αLβ2 constitutive propensity to adhere to ICAM-1 (21Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The substitution of an amide to a hydroxyl side chain (Asn → Ser) hints at the possibility of functional group contribution toward the marked difference in the activity of αLβ2 with β2 Asn329 or Ser329. Therefore, four other variants, αLβ2N329T, αLβ2N329Q, αLβ2N329A, and αLβ2N329D, were generated and tested for their constitutive capacities to adhere to the ICAMs (Fig. 3). All four transfectants constitutively adhered to ICAM-1 even in the absence of Mg/EGTA (Fig. 3A). Similarly, these transfectants adhered constitutively to ICAM-3 (Fig. 3B). The expression levels of αLβ2N329T, αLβ2N329Q, αLβ2N329A, and αLβ2N329D were determined by flow cytometry. The adhesion specificity was assessed by using the αLβ2-specific function-blocking mAb MHM24. It is apparent that the introduction of a hydroxyl group as a result of N329S mutation does not have a primary role in generating a constitutively active αLβ2 because the substitutions N329Q, N329A, and N329D in β2 promoted comparable ICAM-adhesion activity. It is tempting to speculate that the altered binding property of αLβ2N329S is attributed mainly to the loss of the side chain amide group at position 329 of the β2. However, the β2 mutation N329Q, which had a similar activating effect on αLβ2, suggests the requirement of Cβ instead of Cγ amide group at position 329 of the β2 I-like domain to maintain the functional integrity of αLβ2. The LIMBS and MIDAS of the I-like Domain Are Required for the Activating Effect of N329S in I Domain-containing αLβ2—In I domain-containing integrins, the I-like domain allosterically regulates the ligand binding activity of the I domain (16Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (78) Google Scholar). It is reasonable to suggest that structural changes at the locality of β2 N329S are propagated to the ligand-binding face of the I-like domain. This may trigger I-like domain binding of the invariant Glu in the last C-terminal helix of the αL I domain, thus activating I domain ligand binding. Therefore, disrupting the ligand-binding sites of the I-like domain should abrogate the activating signal of N329S. Indeed, transfectants bearing αLβ2 having composite mutations N329S and S116P or D209H failed to adhere to ICAM-1 even in the presence of activating agents (Fig. 4). The adhesion specificity mediated by αLβ2 was demonstrated in all cases by complete abrogation of adhesion in the presence of mAb MHM24. The expression levels of these αLβ2 variants were comparable. Ser116 is the third coordinating residue found in the signature motif DXSXS of the I-like domain MIDAS, and the function disrupting effect of S116P, identified in LAD-1 patient, has been reported (18Hogg N. Stewart M.P. Scarth S.L. Newton R. Shaw J.M. Law S.K. Klein N. J. Clin. Investig. 1999; 103: 97-106Crossref PubMed Scopus (145) Google Scholar). Asp209 is a LIMBS-coordinating residue. Conceivably, the LAD-1 D209H mutation abolished β2 integrin ligand binding capacity (19Mathew E.C. Shaw J.M. Bonilla F.A. Law S.K. Wright D.A. Clin. Exp. Immunol. 2000; 121: 133-138Crossref PubMed Scopus (47) Google Scholar), which corroborates well with the role of LIMBS in stabilizing MIDAS-mediated firm adhesion (5Chen J. Salas A. Springer T.A. Nat. Struct. Biol. 2003; 10: 995-1001Crossref PubMed Scopus (123) Google Scholar). Collectively, these data suggest that the activating effect of N329S is propagated through the ligand-binding site(s) of the β2 I-like domain, which subsequently activates by allostery the αL I domain. Introduction of N339S in β3, Which Corresponds to N329S in β2, Generates a Constitutively Active I-less Integrin αIIbβ3—The Asn at position 329 of integrin β2 is conserved in all integrin β subunits (Fig. 1A). To further demonstrate that the activating signal of Asn mutation is propagated to the ligand-binding site(s) of the I-like domain, we extended the investigation to the I-less integrin αIIbβ3 because the β3 I-like domain participates directly in extrinsic ligand binding (15Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (682) Google Scholar, 37Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1402) Google Scholar). The corresponding Asn339 in β3 (Fig. 1B) was substituted with Ser to generate αIIbβ3N339S. Ser116 and Asp209 of the integrin β2 are also conserved in all integrin β subunits. The corresponding residues in integrin β3 are Ser123 and Asp217. Thus, the MIDAS variant αIIbβ3S123P and the LIMBS variant αIIbβ3D217H were constructed. In addition, the composite variants αIIbβ3N339S/S123P and αIIbβ3N339S/D217H were generated (Fig. 5). Expression levels of αIIbβ3 and variants on transfectants were analyzed by flow cytometry using the β3-specific mAb 7E3. Wild-type αIIbβ3 transfectants adhered avidly to its ligand fibrinogen only in the presence of activating manganese. By contrast, αIIbβ3N339S showed constitutive adhesion to fibrinogen even in the absence of manganese. The specificity of αIIbβ3-mediated adhesion was demonstrated using αIIb-specific function-blocking mAb 10E5. Substitutions S123P and D217H in β3 abolished αIIbβ3-mediated adhesion to fibrinogen. When S123P or D217H was introduced in combination with N339S, it attenuated the activating effect of N339S on αIIbβ3-mediated adhesion to fibrinogen. Theref
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
