Structure of the human MHC-I peptide-loading complex.

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

Calreticulin expression in relation to exchangeable Ca(2+) level that changes dynamically during anthesis, progamic phase, and double fertilization in Petunia.

Calcium (Ca(2+)) plays essential roles in plant sexual reproduction, but the sites and the mechanism of Ca(2+) mobile storage during pollen-pistil interactions have not been fully defined. Because the Ca(2+)-buffering protein calreticulin (CRT) is able to bind and sequester Ca(2+), it can serve as a mobile intracellular store of easily releasable Ca(2+) and control its local concentration within the cytoplasm. Our previous studies showed an enhanced expression of Petunia hybrida CRT gene (PhCRT) during pistil transmitting tract maturation, pollen germination and tube outgrowth on the stigma, gamete fusion, and early embryogenesis. Here, we demonstrate that elevated expression of CRT results in the accumulation of this protein in response to anthesis, pollination, sperm cells deposition within the receptive synergid and fertilization, when the level of exchangeable Ca(2+) changes dynamically. CRT localizes mainly to the endoplasmic reticulum and Golgi compartments in the pistil transmitting tract cells, germinated pollen/tubes, and sporophytic/gametophytic cells of the ovule and corresponds with loosely bound Ca(2+). Additionally, the immunogold research shows, for the first time, highly selective CRT distribution in specific nuclear sub-domains. On the basis of our results, we discuss the possible functions of CRT with respect to the critical role of Ca(2+) homeostasis during key events of the multi-step process of generative reproduction in angiosperms.

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of Rhodnius prolixus Stahl.

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18-24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Partial reversal of transformed fusiform phenotype by overexpression of calreticulin.

Calreticulin, a Ca(2+)-storage and chaperone protein of the ER, has also been shown to affect cell adhesiveness. To examine the effects of differential expression of calreticulin on cellular adhesiveness, we used L fibroblast cell lines stably expressing either elevated or reduced amounts of full length, ER-targeted calreticulin. Overexpression of calreticulin correlates with an increase in adhesiveness of L fibroblasts such that these transformed cells acquire epithelioid morphology and form an epithelial-cell sheet when crowded. Functionally, the "reversal" of transformed phenotype in L fibroblasts differentially overexpressing calreticulin can be accounted for by changes in levels of expression of N-cadherin and vinculin. Structurally, however, although the form and extent of cell-cell contacts in L fibroblasts overexpressing calreticulin mimicked those in normal epithelia, electron microscopical examination revealed that cell-cell junctions formed by these transformed cells bore only superficial resemblance to those of normal epithelia in culture. Our data imply that overexpression of calreticulin, while partially reverses fusiform transformed phenotype is in itself insufficient to re-establish bona fide zonulae adherens in transformed fibroblasts.

Isothermal titration calorimetric study defines the substrate binding residues of calreticulin.

Earlier we established using modeling studies the residues in calreticulin (CRT) important for sugar-binding (M. Kapoor, H. Srinivas, K. Eaazhisai, E. Gemma, L. Ellgaard, S. Oscarson, A. Helenius, A. Surolia, Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone, J. Biol. Chem. 278 (8) (2003) 6194-6200). Here, we discuss the relative roles of Trp-319, Asp-317, and Asp-160 for sugar-binding by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in CNX play important role towards sugar-binding. From the present study we demonstrate that the residue Asp-160 is not involved in sugar-binding, while Asp-317 plays a crucial role. Further, it is also validated that cation-pi interactions of the sugar with Trp-319 dictate sugar-binding in CRT. This study not only defines further the binding site of CRT but also highlights its subtle differences with that of calnexin.

The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain.

Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1-180 fused by a linker to 290-400) and P-domain (189-280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region.

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis.

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.