Calreticulin promotes folding of functional human leukocyte antigen class I molecules in vitro

The assembly of MHC class I molecules with beta(2)-microglobulin and peptides is assisted by the housekeeping chaperones calnexin, calreticulin, and Erp57 and the dedicated accessory protein, tapasin. Tapasin and calreticulin are essential for efficient MHC class I assembly, but their precise action during class I assembly remains to be elucidated. Previous in vitro studies have demonstrated that the lectin calreticulin interacts with monoglucosylated MHC class I heavy chains, whatever their state of assembly with light chains and peptide, and inhibits their aggregation above physiological temperature. We used a soluble single chain HLA-A2/beta(2)-microglobulin molecule, A2SC, to study the effect of calreticulin on the peptide binding capacity of HLA class I molecules. Calreticulin inhibited the formation of A2SC aggregates both when co-expressed in insect cells and during incubations at elevated temperature. Calreticulin dramatically enhanced acquisition of peptide binding capacity when added to denatured A2SC molecules during refolding at 4 degrees C. However, it had no effect on the rapid loss of A2SC peptide binding capacity at physiological temperature. We conclude that calreticulin promotes the folding of HLA class I molecules to a state in which, at low temperature, they spontaneously acquire peptide binding capacity. However, it does not induce or maintain a peptide-receptive state of the class I-binding site, which is likely to be promoted by one or several other components of the class I loading complexes. By being amenable to complementation with additional proteins, the described system should be useful for identification of these components.     

Various properties of the ceruloplasmin receptor, isolated from human erythrocyte membranes

An electrophoretically pure preparation of ceruloplasmin (CP) receptor which retains its ability to bind to CP was isolated from human erythrocyte membranes. It was found that in terms of molecular mass, number and size of spontaneously proteolytic fragments as well as antigenicity, the CP receptor molecule strongly resembles that of CP. A comparative analysis of two-dimensional peptide maps of full tryptic digests of the both protein revealed that about 30% of CP peptides are identical in respect of their electrophoretic and chromatographic mobilities which points to the genetic independence of these proteins. The roles of CP and CP receptor in copper metabolism are discussed.     

The interactions of calreticulin with immunoglobulin G and immunoglobulin Y

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.     

Assessment of roles for calreticulin in the cross-presentation of soluble and bead-associated antigens

Antigen cross-presentation involves the uptake and processing of exogenously derived antigens and their assembly with major histocompatibility complex (MHC) class I molecules. Antigen presenting cells (APC) load peptides derived from the exogenous antigens onto MHC class I molecules for presentation to CD8 T cells. Calreticulin has been suggested to mediate and enhance antigen cross-presentation of soluble and cell-derived antigens. In this study, we examined roles for calreticulin in cross-presentation of ovalbumin using a number of models. Our findings indicate that calreticulin does not enhance in vitro cross-presentation of an ovalbumin-derived peptide, or of fused or bead-associated ovalbumin. Additionally, in vivo, calreticulin fusion or co-conjugation does not enhance the efficiency of CD8 T cell activation by soluble or bead-associated ovalbumin either in wild type mice or in mice lacking Toll-like receptor 4 (TLR4). Furthermore, we detect no significant differences in cross-presentation efficiencies of glycosylated vs. non-glycosylated forms of ovalbumin. Together, these results point to the redundancies in pathways for uptake of soluble and bead-associated antigens.     

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Background  

We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences.     

Bioactive peptides derived from food proteins preventing lifestyle-related diseases

Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy.     

Mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase

Methods for mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase are described with a view to using such data for fine-structure analysis of mutations and/or modifications affecting the protein's electrostatic charge. Peptides generated from [35S]methionine-labeled regulatory subunit were separated by high-resolution two-dimensional gel electrophoresis. Sites of papain cleavage in denatured regulatory subunit were deduced from the kinetics of the appearance, molecular weights, and relative isoelectric points of the fragments produced. These sites and sites of chymotrypsin digestion in the native protein were confirmed by studying peptide overlaps. Carboxy-terminal peptides were identified both by overlaps with cyclic AMP-binding chymotryptic fragments and by their preferential labeling during polysome runoff mediated by pactamycin, an inhibitor of protein initiation. Since peptides containing modifications or mutations that alter protein charge can be identified by shifts in first-dimension isoelectric focusing gel positions, knowledge of fragment endpoints will permit rapid mapping of sites of such alterations by two-dimensional gel analysis of partial proteolytic digests. Such a mapping procedure is inexpensive, can be applied to partially purified proteins or to proteins eluted from polyacrylamide gels, requires only nanogram amounts of the protein of interest, and does not require sequence data to determine relative positions of peptides. Therefore, it provides an attractive alternative to more classical peptide analysis for studying point mutations in cellular proteins of low abundance.     

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies

An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.     

Determination of a putative phosphate-containing peptide in calreticulin.

Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.     

Dimerization and oligomerization of the chaperone calreticulin.

The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 m or SDS concentrations above 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 m at pH below 4.6 or above pH 10, at temperatures above 40 degrees C, or in the presence of high concentrations of organic solvents (25%), conditions that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 m urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 m urea or 1% SDS when present during heating. Comparison of the binding properties of monomeric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured proteins when calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity.     

Lymphocyte suppressive peptides from fibrinogen are derived predominantly from the A alpha chain

Cellular immune responses can elicit local deposition of fibrin at the site of immunologic reactions, as well as the formation of intravascular fibrin in disseminated reactions. The subsequent physiologic proteolysis of fibrinogen and fibrin by plasmin results in small peptides that suppress lymphocyte functions in vitro and in the immune response in vivo. The intramolecular origin of lymphocyte suppressive activity and the proteolytic events responsible for the release of active peptides have been analyzed. Plasmic peptides from the isolated B beta and gamma constituent chains of fibrinogen did not inhibit mitogen-driven responses of human peripheral blood mononuclear cells. In contrast, plasmic digests of the A alpha chain, but not the intact A alpha chain were suppressive. Advanced plasmic digests of fibrinogen and the A alpha chain were suppressive at similar concentrations, suggesting that biological activity is derived predominantly from the A alpha chain. Limited plasmic digests of fibrinogen were fractionated to yield a heat-precipitable 250,000 dalton fragment X and heat-soluble proteolytic products containing fragments derived from the carboxyl-terminal region of the A alpha chain including a 42,000 dalton major A alpha chain derivative. Neither fragment X nor derivatives produced by its additional plasmic proteolysis were suppressive. In contrast, the heat-soluble fraction from limited plasmic cleavage was suppressive, and this activity was enhanced 10-fold by additional plasmic cleavage of this fraction. The isolated 42,000 dalton A alpha chain fragment was devoid of activity, but plasmic digestion of this derivative generated peptides of less than 8000 daltons that inhibited mitogen-stimulated thymidine uptake by lymphocytes. Two synthetic peptides corresponding to A alpha 220-230 and B beta 43-47, peptides with known vasoactive activities, suppressed lymphocyte thymidine uptake at very high concentrations. Based on their maximal yield from plasmic digests of fibrinogen, these two peptides would account for only 1% of the immunosuppressive activity of fibrinogen derivatives. In summary, the results indicate that the suppressive activity of fibrinogen is predominantly derived from the 42,000 dalton carboxyl terminal region of the A alpha chain of the molecule and is not attributable to the known vasoactive peptides. Initial proteolytic release of this region from the core of fibrinogen does not result in suppressive activity, but additional cleavage releases small peptides with the lymphocyte inhibitory function.     

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.     

Analysis of molecular interactions in heat-induced aggregation of a non-inhibitory serpin ovalbumin using a molecular chaperone

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Evidence for complex formation between rabbit lung flavin-containing monooxygenase and calreticulin

Rabbit lung flavin-containing monooxygenase (FMO, EC 1.14.13.8) was denatured, reduced, carboxymethylated, digested with endoproteinase Glu-C or trypsin, and subjected to mass spectrometric analysis. The amino acid sequences of selected peptides were determined by tandem mass spectrometry. Over 90% of rabbit lung FMO was mapped by liquid secondary ion mass spectrometry (LSIMS). The FMO N-terminal amino acid was found to be N-acetylated, and the N-terminal 23 amino acid peptide contained an FAD binding domain consisting of Gly-X-Gly-X-X-Gly. Another peptide was found to contain a NADP+ binding domain consisting of Gly-X-Gly-X-X-Ala. The mapped and/or sequenced peptides were found to be completely consistent with the peptide sequence deduced from the cDNA data and the previously published gas-phase sequencing data. Further mass spectrometry and protein analytical work unambiguously showed that rabbit lung FMO existed in tight association with a calcium-binding protein, calreticulin. Over 68% of rabbit lung calreticulin was mapped by LSIMS. Tandem mass spectrometric and gas-phase sequencing studies provided direct evidence for the identification of the N-terminal and other rabbit lung calreticulin-derived peptide sequences that were identical to other previously reported calreticulins. The complexation of calreticulin to rabbit lung FMO could account for some of the unusual physical properties of this FMO enzyme form.     

Conformational state of disulfide-reduced ovalbumin at acidic pH.

Ovalbumin assumes a highly ordered molten-globule conformation at pH 2.2. To investigate whether or not such structural nature is related to the existence of an intrachain native disulfide bond, the structural characteristics of disulfide-reduced ovalbumin at the acidic pH were compared with those of the native disulfide-intact protein by a variety of analytical approaches. The disulfide-reduced protein was found to assume a partially denatured molten globule-like conformation similar to the disulfide-intact counterpart as analyzed by the CD and intrinsic tryptophan fluorescence spectra and by the binding of a hydrophobic probe of anilino-1-naphthalene-8-sulfonate. The results from size-exclusion chromatography also showed that the disulfide-reduced and disulfide-intact proteins have essentially the same compact, native-like hydrodynamic volume. The disulfide-reduced protein was, however, highly sensitive to proteolysis by pepsin at the acidic pH under the proteolytic conditions in which the disulfide-intact protein was almost completely resistant. Furthermore, on a differential scanning calorimeter analysis the disulfide-reduced protein had an endothermic transition at a much lower temperature (Tm = 48.5 degrees C) than the disulfide-intact protein (Tm = 57.2 degrees C). Taken together, we concluded that the intrachain disulfide bond should not be directly related to the highly ordered molten-globule conformation of ovalbumin, but that its conformational stability depends on the presence of the disulfide bond.     

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.     

Verification by mass spectrometry of the primary structure of human interleukin-2

Proteolytic digests of interleukin-2 from a human leukemic T-cell line produced by Escherichia coli carrying a recombinant DNA were analyzed by fast atom bombardment mass spectrometry. The mass values of intense signals observed in the mass spectrum were consistent with peptides predicted from the nucleotide sequence of cDNA for human interleukin-2, an indication that the protein with the predicted amino acid sequence was produced by E. coli. BrCN and proteolytic digests of interleukin-2 obtained from cultured cells were also examined by fast atom bombardment mass spectrometry. The observed mass values were identical with those from interleukin-2 from E. coli except for that of the NH2-terminal sequence, in which the Thr residue at position 3 was bound to a sugar moiety. The mass spectra of the digests of the two interleukin-2 preparations and synthetic peptides with sequences from 117 to 128 and 121 to 128 predicted from the nucleotide sequence of cDNA for a human interleukin-2 indicated that Cys residues at positions 58 and 105 are linked by a disulfide bond and that the Cys residue at position 125 is free.