Arabidopsis DND2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype

A previous mutant screen identified Arabidopsis dnd1 and dnd2 "defense, no death" mutants, which exhibit loss of hypersensitive response (HR) cell death without loss of gene-for-gene resistance. The dnd1 phenotype is caused by mutation of the gene encoding cyclic nucleotide-gated (CNG) ion channel AtCNGC2. This study characterizes dnd2 plants. Even in the presence of high titers of Pseudomonas syringae expressing avrRpt2, most leaf mesophyll cells in the dnd2 mutant exhibited no HR. These plants retained strong RPS2-, RPM1-, or RPS4-mediated restriction of P. syringae pathogen growth. Mutant dnd2 plants also exhibited enhanced broad-spectrum resistance against virulent P. syringae and constitutively elevated levels of salicylic acid, and pathogenesis-related (PR) gene expression. Unlike the wild type, dnd2 plants responding to virulent and avirulent P. syringae exhibited elevated expression of both salicylate-dependent PR-1 and jasmonate and ethylene-dependent PDF1.2. Introduction of nahG+ (salicylate hydroxylase) into the dnd2 background, which removes salicylic acid and causes other defense alterations, eliminated constitutive disease resistance and PR gene expression but only weakly impacted the HR- phenotype. Map-based cloning revealed that dnd2 phenotypes are caused by mutation of a second CNG ion channel gene, AtCNGC4. Hence, loss of either of two functionally nonredundant CNG ion channels can cause dnd phenotypes. The dnd mutants provide a unique genetic background for dissection of defense signaling.     

GJB2 and GJB6 mutations in 165 Danish patients showing non-syndromic hearing impairment

Thirty-two genes causing non-syndromic hearing impairment (NSHI) have been cloned, including GJB2 and GJB6 encoding the gap junction subunits connexin 26 and connexin 30, respectively. One mutation in GJB2, 35delG, accounts for a large percentage of GJB2 hearing impairment in Southern Europe whereas a considerably lower frequency has been reported from Northern European populations. Recently, a 342-kb deletion implicating GJB6 was found in 22 out of 44 NSHI patients of Spanish origin with only one mutated allele of GJB2. We report the first study of GJB2 and GJB6 mutations in Danish patients with NSHI. We tested 165 individuals and found GJB2 mutations in 16 individuals. The deletion implicating GJB6 was found in two individuals out of 9 heterozygous for GJB2 mutation. Furthermore, we screened 509 unselected samples from the Danish newborn population for the 35delG mutation in GJB2. We found 9 samples heterozygous for 35delG and 11 samples heterozygous for mutations leading to amino acid variants in GJB2 protein. In conclusion, our data are in accordance with results from other Northern European populations. Furthermore, our data on the GJB6 deletion suggest that routine screening for this deletion could help to explain hearing impairment in some Northern European NSHI patients heterozygous for a mutation in GJB2.     

Molecular genetic analysis of severe protein C deficiency

Severe protein C deficiency is a rare, early onset, venous thrombotic condition that is inherited as an autosomal recessive trait. The protein C (PROC) genes of nine unrelated individuals with severe protein C deficiency were sequenced yielding a total of 13 different lesions. Eight of these were novel, including a gross gene deletion, three missense mutations, two micro-deletions, a splicing mutation and a single base-pair substitution in the HNF-3 binding site in the PROC gene promoter. Evidence for the pathogenicity of the mutations detected was obtained by molecular modelling, in vitro splicing assay and reporter gene assay. Neither the plasma protein C activity level nor the nature of the PROC gene lesions detected were found to be a good prognostic indicator of the age of onset or clinical severity of thrombotic symptoms. Other factors may thus complicate the relationship between genotype and clinical phenotype. Indeed, in two patients, the inheritance of either one or two Factor V Leiden alleles in addition to two PROC gene lesions could have served to precipitate the thrombotic events. No association was however apparent between clinical severity and the possession of a particular promoter polymorphism genotype. Despite the absence of a clear genotype-phenotype relationship, the molecular genetic analysis of the severe recessive form of protein C deficiency potentiates both the counselling of affected families and the provision of antenatal exclusion diagnosis.     

Probing the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions of specific domains to signaling function

Flagellin sensing2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu receptor that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.     

Monoclonal TCR-redirected tumor cell killing

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.     

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.     

Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, alphaSMA, PDGFR-beta). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ(+) PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.     

Mice Lacking the Extracellular Matrix Protein WARP Develop Normally but Have Compromised Peripheral Nerve Structure and Function

WARP is a recently identified extracellular matrix molecule with restricted expression in permanent cartilages and a distinct subset of basement membranes in peripheral nerves, muscle, and the central nervous system vasculature. WARP interacts with perlecan, and we also demonstrate here that WARP binds type VI collagen, suggesting a function in bridging connective tissue structures. To understand the in vivo function of WARP, we generated a WARP-deficient mouse strain. WARP-null mice were healthy, viable, and fertile with no overt abnormalities. Motor function and behavioral testing demonstrated that WARP-null mice exhibited a significantly delayed response to acute painful stimulus and impaired fine motor coordination, although general motor function was not affected, suggesting compromised peripheral nerve function. Immunostaining of WARP-interacting ligands demonstrated that the collagen VI microfibrillar matrix was severely reduced and mislocalized in peripheral nerves of WARP-null mice. Further ultrastructural analysis revealed reduced fibrillar collagen deposition within the peripheral nerve extracellular matrix and abnormal partial fusing of adjacent Schwann cell basement membranes, suggesting an important function for WARP in stabilizing the association of the collagenous interstitial matrix with the Schwann cell basement membrane. In contrast, other WARP-deficient tissues such as articular cartilage, intervertebral discs, and skeletal muscle showed no detectable abnormalities, and basement membranes formed normally. Our data demonstrate that although WARP is not essential for basement membrane formation or musculoskeletal development, it has critical roles in the structure and function of peripheral nerves.WARP (von Willebrand A domain-related protein) is a recently described member of the von Willebrand factor type A domain (VWA² domain) superfamily of extracellular matrix (ECM) molecules, adhesion proteins, and cell surface receptors (for review, see Ref. 1). The WARP protein is encoded by the Vwa1 (von Willebrand factor A domain-containing 1) gene and comprises a single N-terminal VWA domain containing a putative metal ion-dependent adhesion site (MIDAS) motif, two fibronectin type III repeats, and a unique C-terminal domain that contributes to WARP multimer formation (2, 3). Like many other VWA domain-containing extracellular molecules, WARP was predicted to participate in protein-protein interactions and in the formation of supramolecular structures. Recently WARP has been shown to interact with the heparan sulfate proteoglycan perlecan (3), and in the present study we identify type VI collagen as a ligand for WARP.WARP has a restricted distribution in developing cartilage tissues, where it is expressed at sites of joint cavitation and articular cartilage formation rather than cartilage structures that will undergo endochondral ossification (3). In adult tissues, WARP is highly restricted to the chondrocyte pericellular matrix in articular cartilage and fibrocartilages, where it co-localizes with perlecan and collagen VI (3). Several of the major basement membrane components have been found in the chondrocyte pericellular matrix, suggesting that this structure may be the functional equivalent of a basement membrane in cartilage tissues (4). Consistent with this hypothesis, recent data from our laboratory have demonstrated that WARP is a component of the basement membrane in a limited subset of tissues including the apical ectodermal ridge, the endomysium surrounding muscle fibers, the vasculature of the central nervous system, and the endoneurium of peripheral nerves (5). The principal components of basement membranes are type IV collagen, laminins, nidogens, and proteoglycans including perlecan; however, the composition, structure, and biological properties of basement membranes can differ considerably between different tissues (6, 7). Different isoforms of the major components contribute to the heterogeneity of basement membranes, but the contribution of quantitatively minor components to particular subtypes of basement membranes and their interactions with surrounding cells and ECM structures are poorly understood (8, 9).We, therefore, have generated mice with a targeted disruption of the WARP locus to determine the consequences of WARP deficiency on skeletal development and basement membrane formation. The homozygous null mice are viable, fertile, and do not exhibit overt abnormalities compared with wild type littermates. Neurological testing revealed that WARP-null mice exhibit a delayed response to acute painful stimulus and a disturbance in fine motor coordination, although general motor function is not impaired. Consistent with these findings, immunohistochemical analysis of peripheral nerves from WARP-null mice revealed that the collagen VI microfibrillar matrix was severely reduced and mislocalized compared with wild type mice. Furthermore, electron microscopic examination of the sciatic nerve demonstrated a reduction in the collagen I ECM and the unusual partial fusing of the basement membranes of neighboring axons. These data suggest an important role for WARP in organizing the peripheral nerve ECM and provides evidence for tissue-specific differences in the role of WARP in the assembly and/or integration of the ECM. In addition, our studies provide further evidence for the critical role of ECM structure and organization in nerve function.     

Germ Line-governed Recognition of a Cancer Epitope by an Immunodominant Human T-cell Receptor

CD8⁺ T-cells specific for MART-1-(26–35), a dominant melanoma epitope restricted by human leukocyte antigen (HLA)-A*0201, are exceptionally common in the naive T-cell repertoire. Remarkably, the TRAV12-2 gene is used to encode the T-cell receptor α (TCRα) chain in >87% of these T-cells. Here, the molecular basis for this genetic bias is revealed from the structural and thermodynamic properties of an archetypal TRAV12-2-encoded TCR complexed to the clinically relevant heteroclitic peptide, ELAGIGILTV, bound to HLA-A*0201 (A2-ELA). Unusually, the TRAV12-2 germ line-encoded regions of the TCR dominate the major atomic contacts with the peptide at the TCR/A2-ELA interface. This “innate” pattern of antigen recognition probably explains the unique characteristics and extraordinary frequencies of CD8⁺ T-cell responses to this epitope.Malignant melanoma is responsible for 75% of all skin cancer-related deaths worldwide, and the global incidence is rising. The MART-1 (1) protein, also known as Melan-A (2), is expressed by virtually all fresh melanoma tumor specimens and elicits natural CD8⁺ T-cell responses (3, 4) that can lead to spontaneous disease regression (reviewed in Ref. 5). Consequently, CD8⁺ T-cell responses directed against the MART-1 protein have been investigated extensively (reviewed in Refs. 2, 6, and 7), and heteroclitic forms of the dominant MART-1-(26–35) peptide epitope (8, 9), which is restricted by human leukocyte antigen (HLA)-A*0201, are currently being used in a number of clinical trials (10–12). In recent developments, adoptive T-cell therapy directed against the MART-1 protein has been used to mediate cancer regression in ∼50% of late stage melanoma patients (13). However, these approaches have not proved to be universally effective, and there remains considerable scope for improvement. In order to design more effective immune-based therapies against the MART-1 protein, it is essential to understand the precise molecular rules that govern the interaction between T-cell receptors (TCRs)⁶ and the HLA-A*0201·MART-1-(26–35) complex. Previous structural studies of human TCR/peptide major histocompatibility complex (pMHC) interactions (14–16) indicate that specific regions of the TCR have different roles during antigen engagement; thus, the germ line-encoded complementarity-determining region 1 and 2 (CDR1 and -2) loops contact mainly the conserved helical region of the MHC surface, and the more variable somatically rearranged CDR3 loops contact mainly the antigenic peptide. Dissecting the nature of these contacts, which have been shown to be highly variable for individual TCR/pMHC interactions (17–19), is an important step toward understanding the principles of antigen recognition and for the development of improved T-cell vaccines (20). However, the current data base of human TCR·pMHC complexes reported in the literature is limited (∼16), compared with >100 antibody-antigen structures. This has made it difficult to ascertain whether there are conserved binding modes for TCR/pMHC interactions dictated by a number of specific contacts or whether there are potentially unlimited numbers of TCR docking orientations dependent on the nature of individual recognition events. Furthermore, there are no examples to date of human TCR·pMHC class I structures in which the bound peptide is a decamer; this represents a substantial deficiency in our current knowledge, given the preponderance with which decamer peptides are processed, presented, and recognized. The low number of TCR·pMHC complex structures solved to date reflects technical difficulties inherent in the production of soluble TCR and pMHC molecules that retain stability and challenges related to the crystallization of complexes with relatively low binding affinities (K_D = 0.1–500 μm) (21, 22). In general, TCRs specific for tumor-derived epitopes bind in the weaker range of TCR/pMHC affinities (21). This obstacle to the generation of high quality co-complex crystals is underscored by the fact that only one other tumor-specific human TCR·pMHCI complex structure has been documented previously (23).In this study, we expressed a soluble TCR (MEL5) specific for ELAGIGILTV, the common MART-1-(26–35) heteroclitic peptide, complexed to HLA-A*0201 (A2-ELA). Notably, HLA-A*0201 is the most common HLA allele in the human population (24). The CDR1 and CDR2 loops of this TCR are encoded by the TRAV12-2 and TRBV30 genes (International Immunogenetics (IMGT) nomenclature). Interestingly, the TRAV12-2 gene is expressed in the vast majority of CD8⁺ T-cell populations specific for HLA-A*0201·MART-1-(26–35) across multiple individuals (25, 26). To resolve the enigma of the dominant TRAV12-2 gene and determine the molecular characteristics that govern CD8⁺ T-cell recognition of the HLA-A*0201·MART-1-(26–35) antigen, we performed a biophysical, thermodynamic, and structural analysis of MEL5 TCR binding to A2-ELA. The data provide a molecular basis for biased TCR gene product selection in the CD8⁺ T-cell response to HLA-A*0201·MART-1-(26–35) and indicate that pMHC antigens can be subject to “innate-like” binding modes within adaptive immune responses.