Paul Eggleton

The ins and outs of calreticulin: from the ER lumen to the extracellular space

Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca(2+)-binding protein of the sarcoplasmic reticulum, the appreciation of its importance has grown, and it is now recognized to be a multifunctional protein, most abundant in the endoplasmic reticulum (ER). The protein has well-recognized physiological roles in the ER as a molecular chaperone and Ca(2+)-signalling molecule. However, it has also been found in other membrane-bound organelles, at the cell surface and in the extracellular environment, where it has recently been shown to exert a number of physiological and pathological effects. Here, we will focus on these less-well-characterized functions of calreticulin.

Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses.

We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.

Calreticulin: non-endoplasmic reticulum functions in physiology and disease

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT‐driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT‐null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER‐resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.—Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.‐M., Michalak, M., Murphy‐Ullrich, J. E. Calreticulin: non‐endoplamic reticulum functions in physiology and disease. FASEB J. 24, 665–683 (2010). www.fasebj.org

Thrombospondin Mediates Focal Adhesion Disassembly through Interactions with Cell Surface Calreticulin

Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17–35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, binds a hep I affinity column, and neutralizes thrombospondin/hep I-mediated focal adhesion disassembly. Calreticulin on the cell surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-activated cell sorting analyses. Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calreticulin and preincubation of thrombospondin/hep I with glutathioneS-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.

A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins

Calreticulin was recently identified as a hookworm Necator americanus allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin‐mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins αIIb and α5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase‐polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host–parasite relationship.

Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers

A simple, rapid and economical method for the isolation of polymorphonuclear leucocytes (PMNs) from whole blood is compared with dextran and dextran/Lymphoprep gradient techniques. The method eliminates the use of dextran and density gradient polymers such as Ficoll which have been shown to affect PMNs adversely. The technique is based on the lysis of red cells with isotonic ammonium chloride solution followed by differential centrifugation to separate the PMNs. This method gave a PMN yield of 73% (SD +/- 3.5) and a purity of 78% (SD +/- 2.5). Both morphology and functional activity were preserved, as assessed by bacterial phagocytosis and killing, chemotaxis, polarising response, superoxide production and adherence. In contrast, the dextran and dextran/Lymphoprep techniques gave yields of 50% and 15% with purities of 78% and 91% respectively. In a series of 14 PMN isolations, the differential centrifugation method gave an average yield of 63% with an average purity of 83%.

The Anti-adhesive Activity of Thrombospondin Is Mediated by the N-terminal Domain of Cell Surface Calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17–35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but notcrt −/− cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1–43 but not a construct of aa 1–31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19–36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19–36 (glutathione S-transferase-CRTΔhep I) failed to restore responsiveness to hep I in crt −/−cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.

Lung surfactant proteins involved in innate immunity.

The two lung surfactant collectins, surfactant protein (SP)-A and SP-D, are involved in host defence against infectious and allergenic agents via enhancement of killing and clearance by macrophages and neutrophils. Recent gene-knockout, protein engineering and physiological studies have emphasised the roles that SP-A and SP-D play in acute inflammatory responses.

C1q—how many functions? How many receptors?

C1, the first component of the classical pathway of complement activation is a complex of three proteins called C1q, C1r and C1s. Normally, C1q binding to aggregated IgG molecules results in activation of the classical pathway of complement. However, C1q has a number of other observed functions, not directly related to complement, that could be mediated by recently identified binding proteins acting as cell-surface receptors or soluble modulators of C1q-mediated functions. This article discusses the various activities of C1q and the evidence that these functions might be influenced by both membrane-bound and soluble C1q-binding proteins.

Consequence of neo-antigenicity of the ‘altered self’

Post-translational modifications play a central role in determining the function of proteins. Such protein modifications come in a great variety of guises, and include phosphorylation, proteolysis, glycosylation, citrullination and oxidative modifications. In relation to inflammatory autoimmune diseases, some post-translational modifications appear to result in the generation of new antigens, and hence autoantibodies. Examples include: the induction of peptide immunogenicity by the spontaneous conversion of aspartic acid residues to isoaspartic acid; granzyme B-mediated cleavage of SLE autoantigens; the oxidative modification--on the surface of apoptotic cells--of lipids and proteins, rendering them immunogenic; and the presence of antibodies to oxidatively modified type II collagen and C1q in RA and SLE patients, respectively. The measurement of autoantibodies to citrullinated proteins has been verified as a very useful diagnostic tool in RA. Proteomics techniques, in principle, allow the detection of all types of in vivo protein modifications, and the increasing application of such technologies to the study of rheumatological diseases will further our understanding of autoantigenicity.