Development of comprehensive functional genomic screens to identify novel mediators of osteoarthritis.

OBJECTIVE: The aim of this study was to develop high-throughput assays for the analysis of major chondrocyte functions that are important in osteoarthritis (OA) pathogenesis and methods for high-level gene expression and analysis in primary human chondrocytes. METHODS: In the first approach, complementary DNA (cDNA) libraries were constructed from OA cartilage RNA and full-length clones were selected. These cDNAs were transferred into a retroviral vector using Gateway Technology. Full-length clones were over-expressed in human articular chondrocytes (HAC) by retroviral-mediated gene transfer. The induction of OA-associated markers, including aggrecanase-1 (Agg-1), matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), collagen IIA and collagen X was measured by quantitative real-time polymerase chain reaction (QPCR). Induction of a marker gene was verified by independent isolation of 2-3 clones per gene, re-transfection followed by QPCR as well as nucleotide sequencing. In the second approach, whole cDNA libraries were transduced into chondrocytes and screened for chondrocyte cluster formation in three-dimensional agarose cultures. RESULTS: Using green fluorescent protein (eGFP) as a marker gene, it was shown that the retroviral method has a transduction efficiency of >90%. A total of 40 verified hits were identified in the QPCR screen. The first set of 19 hits coordinately induced iNOS, COX-2, Agg-1 and MMP-13. The most potent of these genes were the tyrosine kinases Axl and Tyro-3, receptor interacting kinase-2 (RIPK2), tumor necrosis factor receptor 1A (TNFR1A), fibroblast growth factor (FGF) and its receptor FGFR, MUS81 endonuclease and Sentrin/SUMO-specific protease 3. The second set of seven hits induced both Agg-1 and MMP-13 but none of the other markers. Five of these seven genes regulate the phosphoinositide-3-kinase pathway. The most potently induced OA marker was iNOS. This marker was induced 20-500 fold by seven genes. Collagen IIA was also induced by seven genes, the most potent being transforming growth factor beta (TGFbeta)-stimulated protein TSC22, vascular endothelial growth factor (VEGF) and splicing factor 3a. This screening assay did not identify inducers of collagen X. The second chondrocyte cluster formation screen identified 14 verified hits. Most of the genes inducing cluster formation were kinases. Additional genes had not been previously known to regulate chondrocyte cluster formation or any other chondrocyte function. CONCLUSIONS: The methods developed in this study can be applied to screen for genes capable of inducing an OA-like phenotype in chondrocytes on a genome-wide scale and identify novel mediators of OA pathogenesis. Thus, coordinated functional genomic approaches can be used to delineate key genes and pathways activated in complex human diseases such as OA.

Cytokines in Drosophila hematopoiesis and cellular immunity.

Hematopoiesis is a complex, multistep process in which progenitor cells undergo distinct cellular changes of proliferation and differentiation to give rise to mature blood cells in circulation. Many of the genetic and molecular events that drive these changes have been characterized in mammals, frogs, and zebra fish, and more recently in the insect model system Drosophila melanogaster. Blood cells in Drosophila are actively involved in fighting infections and the cellular immune responses are intimately tied to the process of hematopoiesis. In this article, we briefly review the fundamental similarities in Drosophila and mammalian hematopoiesis and highlight the potential roles of four cytokines/growth factors in Drosophila hematopoiesis and cellular immunity.

A splice variant of beta-secretase deficient in the amyloidogenic processing of the amyloid precursor protein.

beta-Secretase (BACE) initiates the amyloidogenic processing of the amyloid precursor protein leading to the generation of the beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE is a type I transmembrane aspartyl protease of 501 amino acids. Here we describe a novel BACE mRNA lacking 132 base pairs that is expressed in the pancreas but not in the brain. Sequence alignment indicates that the deleted fragment matches the terminal two-thirds of exon 3. The new BACE variant is short of a 44-amino acid region located between the two catalytic aspartyl residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains in a proenzymatic and endoglycosidase H-sensitive state, suggesting that its transport along the secretory pathway is blocked at the level of the endoplasmic reticulum. Notably, this novel form of BACE does not contribute to the processing of the amyloid precursor protein. Our findings suggest that tissue-specific splicing of the BACE mRNA may explain the observation that in the human pancreas robust transcription of the BACE gene does not translate into recovered enzymatic activity.

Mutational analysis of the CD6 ligand binding domain.

CD6 belongs to the scavenger receptor cysteine-rich protein superfamily (SRCRSF), which includes a large number of cell surface proteins. The extracellular region of CD6 is composed of three SRCR domains. The membrane proximal SRCR domain of CD6 (CD6D3) specifically binds activated leukocyte cell adhesion molecule (ALCAM), a cell surface protein which is a member of the immunoglobulin superfamily (IgSF). CD6-ligand interactions have been implicated in immune cell adhesion, T cell maturation and the regulation of T cell activation. We tested 13 CD6D3 mutant proteins for binding to ALCAM and a panel of conformationally sensitive anti-CD6D3 monoclonal antibodies (mAbs). CD6D3 residues were classified according to their importance for structural integrity and ligand binding. The results were analyzed in the light of SRCR domain sequence comparison. A number of residues critical for ligand binding or important for structural integrity cluster in the C-terminal region of CD6D3 which is not conserved in other SRCR proteins.

Identification of residues in CD6 which are critical for ligand binding.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics.

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell-APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 0.43 s-1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate-dynamic T cell-APC contacts and to facilitate scanning of APC for antigen.

Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements.

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones.

Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. We reasoned that small molecules that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compounds, 4A,5,8,8A-tetrahydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by alpha-fusion peptide antisera. Several derivatives of this compound, including both benzoquinones and hydroquinones, also showed inhibition. The most effective compounds tested have IC50S between 1 and 20 microM. Representative compounds also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.

Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.

Electron microscopic atlas of the simian cochlea.

An electron microscopic atlas of simian cochlear duct structures is presented with the hope that it may help to clarify certain questions of structural-functional relationships. Emphasis is placed on structures directly involved in sensory transduction and in synaptic transmission, at afferent and efferent nerve fiber terminals. A brief discussion is included as an introduction to the possibilities of interpretation of electron microscopic data. Orientation of electron micrographs is assisted by means of numerical coding on light microscopic photographs, and by means of a diagram which combines electron microscopic and light microscopic data.

Presynaptic bodies of auditory hair cells in Old World monkeys.

Presynaptic bodies of auditory hair cells of Old World monkeys are separately differentiated in inner, as contrasted with outer, hair cells. The pre-synaptic bodies of outer cells are spherical and of variable electron density, and are thus similar to those of the labyrinth of vertebrates from fish to man. The difficulty in finding them, as compared with the relative ease of finding the presynaptic bodies of inner hair cells, suggests either that they are not present in all outer hair cells or that they undergo a regression-reconstitution cycle. The presynaptic bodies of simian inner hair cells are almost always ring-shaped. The few exceptions reinforce the impression of a later evolutionary development of the inner hair cell system. In any event, our findings serve to reemphasize the remarkable differentiation of outer and inner hair cell systems, and to deepen the mystery of their separate role in audition.

Denervation study of synapses of organ of Corti of old world monkeys.

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer hair-cell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of different axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.

Synapses involving auditory nerve fibers in primate cochlea.

The anatomical mechanisms for processing auditory signals are extremely complex and incompletely understood, despite major advances already made with the use of electron microscopy. A major enigma, for example, is the presence in the mammalian cochlea of a double hair cell receptor system. A renewed attempt to discover evidence of synaptic coupling between the two systems in the primate cochlea, postulated from physiological studies, has failed. However, in the outer spiral bundle the narrow and rigid clefts seen between pairs of presumptive afferent fibers suggest the possibility of dendro-dendritic interaction confined to the outer hair cell system. The clustering of afferent processes within folds of supporting cells subjacent to outer hair cells is in contrast to the lack of such close associations in the inner hair cell region. The difference reinforces the suggestion of functional interaction of some sort between the outer hair cell afferent nerve processes.

Chronic progressive poliomyelitis secondary to vaccination of an immunodeficient child.

We investigated an immunodeficient child in whom chronic progressive poliomyelitis developed after she had received live oral poliovirus vaccine. Poliovirus, Type II, was isolated from throat and stool during life and from several sites within the brain at autopsy. The brain isolate was classified as vaccine-like on the basis of temperature sensitivity and antigenic markers. However, in the monkey neurovirulence test, the brain isolate produced moderately severe lesions throughout the spinal cord and brainstem and appeared nonvaccine-like. Thus, the brain isolate demonstrated a dissociation between the antigenic and neurovirulence markers. Our observations suggest that, under unusual circumstances, such as immunodeficiency, attenuated poliovirus can produce a chronic progressive neurologic disease. This case also emphasizes the need to diagnose immunodeficiency as early as possible, so that live-virus vaccines will not be administered.

Poliomyelitis and the sources of useful knowledge.

Dr. Richard Ross during his introduction observed that Dr. Bodian made three contributions to the study of poliomyelitis which laid the groundwork essential to the ultimate triumph over the disease by immunization. The first was the identification of three types of poliovirus. This was absolutely basic to the development of a vaccine. The second was the identification of the viremia which occurred before the paralysis began and thus, indicated how it would be possible for an immune response to be effective. The third important contribution was the demonstration that immunity could be developed in primates with very low antibody levels.