Surface geochemistry of the clay minerals

Clay minerals are layer type aluminosilicates that figure in terrestrial biogeochemical cycles, in the buffering capacity of the oceans, and in the containment of toxic waste materials. They are also used as lubricants in petroleum extraction and as industrial catalysts for the synthesis of many organic compounds. These applications derive fundamentally from the colloidal size and permanent structural charge of clay mineral particles, which endow them with significant surface reactivity. Unraveling the surface geochemistry of hydrated clay minerals is an abiding, if difficult, topic in earth sciences research. Recent experimental and computational studies that take advantage of new methodologies and basic insights derived from the study of concentrated ionic solutions have begun to clarify the structure of electrical double layers formed on hydrated clay mineral surfaces, particularly those in the interlayer region of swelling 2:1 layer type clay minerals. One emerging trend is that the coordination of interlayer cations with water molecules and clay mineral surface oxygens is governed largely by cation size and charge, similarly to a concentrated ionic solution, but the location of structural charge within a clay layer and the existence of hydrophobic patches on its surface provide important modulations. The larger the interlayer cation, the greater the influence of clay mineral structure and hydrophobicity on the configurations of adsorbed water molecules. This picture extends readily to hydrophobic molecules adsorbed within an interlayer region, with important implications for clay–hydrocarbon interactions and the design of catalysts for organic synthesis.

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Vα transcripts (Vα1 and Vα2) and one TCR Vβ transcript (Vβ13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Vα1Vβ13 combination specifically recognized both the HLA-A2+ and HLA-DR1+ target cells, whereas the Vα2Vβ13 combination did not result in a TCR on the cell surface. Thus a single TCRαβ complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.

The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Real-time detection of the surface delivery of newly synthesized membrane proteins.

Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting.

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.

Protein-protein interactions in the rigor actomyosin complex.

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops. The model building approach used with actomyosin is applicable to other multicomponent assemblies of biological interest and is a powerful method for revealing molecular interactions and providing insights into the mode of action of the assemblies.

Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex.

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man.