Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.     

Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.     

Cloning of human DECTIN-1, a novel C-type lectin-like receptor gene expressed on dendritic cells

We identified a new Ca2+-dependent lectin-like receptor gene, DECTIN-1 (HGMW-approved symbol CLECSF12), the human orthologue of mouse Dectin-1, coding for a putative type II transmembrane glycoprotein with an extracellular C-type lectin-like domain. The gene structure and two alternative spliced forms of DECTIN-1 are described. The DECTIN-1 gene was localized in the natural killer gene complex on human Chromosome 12p12.3-p13.2, between OLR1 and CD94 (position 21.8 cM on the genetic map). The DECTIN-1 gene is highly expressed at the mRNA level in dendritic cells and is not further up-regulated during the maturation of these cells with tumor necrosis factor-alpha. The DECTIN-1 gene therefore represents a novel human member of the C-type lectin-like receptor gene family preferentially expressed in dendritic cells.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes

We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3(+) dendritic cells and on a small subset of CD14(+)CD16(-) monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.     

Crystal structure of KD93, a novel protein expressed in human hematopoietic stem/progenitor cells

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked alpha/beta domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.     

Crystal structure of human CD38 extracellular domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.     

CD44 is a major E-selectin ligand on human hematopoietic progenitor cells

E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.     

The production of soluble C-type lectin-like receptor 2 is a regulated process

C-type lectin-like receptor 2 (CLEC-2) is a newly identified type II transmembrane protein belonging to the C-type lectin family molecules, which acts as a cell-surface receptor for snake venom toxin rhodocytin and tumor antigen podoplanin. We previously demonstrated that the full-length mouse CLEC-2 (mCLEC-2) can be cleaved into soluble form. Elevated levels of soluble forms of membrane proteins in circulating blood may reflect increased expression of membrane proteins and disease activities. In the present study, we clarified the domain and sites contributing to the production of soluble mCLEC-2. The shedding process can be positively regulated by protein kinase C (PKC). Moreover, we explored the possibility that human CLEC-2 (hCLEC-2) may also be proteolyticly cleaved and released as a soluble form. We have observed that the production of soluble hCLEC-2 could be induced by phorbol ester (PMA) in cells stably transfected with hCLEC-2 cDNA. Further studies may explore therapeutic and diagnostic applications of soluble hCLEC-2 in platelet-related diseases.     

Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial alpha-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.     

Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.     

Normal and leukemic CD34-negative human hematopoietic stem cells

Considerable progress has been made in recent years in purifying human and murine hematopoietic stem cells. The essential marker identified is the sialomucin CD34, which is expressed on primitive cells and downregulated as they differentiate into more abundant mature cells. CD34 is not unique to stem cells, however, as it is also expressed on clonogenic progenitors and some endothelial cells. Nevertheless, all clinical and experimental protocols are targeted to CD34+ cells enriched by a variety of selection methods. Recent studies in both the murine and human systems have indicated that some stem cells capable of multilineage repopulation do not express detectable levels of cell surface CD34. These studies challenge the dogma that all human repopulating cells are found in the CD34+ subset. However, the precise relationship between CD34- and CD34+ stem cells is still not well understood. In this review, the results on the discovery of the CD34- repopulating cell are summarized and the impacts this discovery may have, both clinically and in our understanding of the organization of the human hematopoietic system, are examined.     

Identification and characterization of a spliced C-type lectin-like gene encoded by rat cytomegalovirus

The English isolate of rat cytomegalovirus (RCMV) encodes a 20-kDa protein with a C-type lectin-like domain that is expressed in the delayed-early and late phases of the viral replication cycle. Genomic sequence analysis of the restriction fragment KpnR of RCMV revealed significant homology to several C-type lectin-containing molecules implicated in natural killer (NK) and T-cell interactions, as well as genes from four poxviruses and African swine fever virus. The gene is spliced into five exons and shows a splicing pattern with exon boundaries similar to those observed in the human differentiation antigen CD69. The cap site of the gene was mapped by RNase protection, 5' rapid amplification of cDNA ends, and primer extension experiments. This analysis demonstrated that the core promoter of the RCMV lectin-like gene contains a GATA rather than a TATA box. Splicing patterns were confirmed with isolates from an infected-cell cDNA library. A unique aspect of the protein is that its translation is not initiated by the canonical methionine but rather by alanine. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted. Replication in tissue culture was similar to that of wild-type virus.     

CD57, a marker for B-cell activation and splenic ellipsoid-associated reticular cells of the chicken

We have demonstrated that the ellipsoid-associated reticular cells of chicken spleen express CD57, a marker for B-cell activation. These cells are characterised by their spindle-shaped morphology, tissue distribution and the absence of certain leucocyte-specific markers. They are phagocytotic and possess high endogenous non-specific esterase activity. Previous reports failed to detect CD57 expression on ellipsoid-associated reticular cells, probably because the tissue sections were differently treated before immunohistochemistry. CD57 is also expressed by a small number of T-cells in the spleen and the caecal tonsils. This number is highly variable between individual chickens depending on the activation state of the immune system. Moreover, CD57 is expressed by bursal lymphocytes (90% or more) but not by B-cells of the peripheral blood. More interestingly, we have been able to discriminate and quantify three B-cell populations of the secondary lymphoid organs, i.e. resting B-cells, germinal centre B-cells and plasma cells, based on their expression levels of CD57 and Bu-1 (a pan B-cell marker). Thus, CD57 should be considered as a B-cell activation marker, rather than as a marker for bursal B-cells; it is also a valuable marker for the immunohistochemical study of ellipsoid-associated reticular cells of chicken spleen.     

CD54 is a surrogate marker of antigen presenting cell activation

There is no single universally accepted hallmark of antigen presenting cell (APC) activation. Instead a variety of methods are used to identify APCs and assess their activation state. These activation measures include phenotypic methods [e.g., assessing the increased expression of surface markers such as major histocompatability (MHC) class II] and functional assays (e.g., evaluating the enhanced ability to take up and process antigen, or stimulate naïve T cells). Sipuleucel-T is an investigational autologous active cellular immunotherapy product designed to stimulate a T cell immune response against human prostatic acid phosphatase (PAP), an antigen highly expressed in prostate tissue. Sipuleucel-T consists of peripheral blood mononuclear cells (PBMCs), including activated APCs displaying epitopes of PAP. In order to develop a robust reproducible potency assay that is not hampered by MHC restriction we have developed a method to simply assess the biological activation of antigen presenting cells (APCs). In the course of sipuleucel-T characterization, we analyzed various phenotypic and functional parameters to define the activation state of APCs obtained from peripheral blood. Flow cytometric assays revealed that CD54+ cells are responsible for antigen uptake, and that expression of CD54 predominantly localizes to APCs. Costimulation, as measured by an allogeneic mixed lymphocytic reaction (alloMLR) assay, showed that activity was restricted to the CD54+ cell population. Similarly, CD54+ cells harbor all of the PAP-specific antigen presentation activity, as assayed using a PAP-specific HLA-DRβ1-restricted T cell hybridoma. Finally we show that CD54 expression is substantially and consistently upregulated on APCs during culture with a GM-CSF fusion protein, and that this upregulation activity can be quantified. Thus these data support the use of CD54 upregulation as a surrogate for assessing human APC activation and validates its utility as a potency measure of sipuleucel-T.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.