Cover Picture: Eur. J. Immunol. 2/11

The cover image of this issue consists of the immunohistochemical staining of μ‐calpain in a healthy human kidney and is taken from Letavernier et al. (pp. 473–484). In this article, the authors analyse the role of calpains in acute allograft rejection. The authors demonstrate that μ‐calpain expression is increased in human transplant kidneys undergoing acute or chronic rejection, mainly in infiltrating T cells. The authors further show that calpain inhibition delays allograft rejection. Interestingly, this delay in allograft rejection is associated with a limitation in allograft infiltration by T cells that is due to the slowing down in T‐cell migration rather than proliferation.     

Characterization and comparative expression of zebrafish calpain system genes during early development

The classic calpain system has been implicated in regulating a variety of cellular processes including cell adhesion, migration, and intracellular signaling; however, little is known regarding the function of this system in vivo. Two heterodimeric Ca(2+)-dependent cysteine proteases, mu-calpain (CAPN1) and m-calpain (CAPN2), and the endogenous inhibitor calpastatin (CAST) comprise the classic/ubiquitous calpain system in mammals. Recently, knockout of two murine classic calpain genes, Capn2 and Capn4/Capns1, revealed that components of the classic system are indispensable for preimplantation development. We identified four classic calpain catalytic subunit genes (capn1a, 1b, 2a, 2b), two regulatory subunit genes (capns1a, 1b), and calpastatin (cast) from the zebrafish. Our data suggest that the components of the classic mammalian system are both conserved and expanded in the teleost lineage. In contrast to the classic/ubiquitous mammalian system, zebrafish calpain system genes acquire unique, tissue-specific patterns of expression within the first 2 days of development.     

The calpain system and diabetes

Diabetes mellitus is recognized as a clinical syndrome that is characterized by hyperglycemia due to deficiency of insulin. The global prevalence of diabetes has been estimated to increase from 4% (1995) to 5.4% by the year 2025. Insulin dependent diabetes mellitus (IDDM/Type-1) in human, generating hyperglycemia due to insulin deficiency as a consequence of destructing beta cells in the pancreatic islets. Non-insulin dependent diabetes mellitus (NIDDM/Type-II), is a multifactorial, exact biochemical and genetic defect which has not yet been elucidated completely. Calpains seem to play a role in NIDDM and IDDM. Positional cloning experiments revealed that there is a NIDDM susceptibility to calpain 10 (CAPN10). Increased calpain activity and leukocyte trafficking were noticed in the microcirculation in ZDF (Zuker diabetic fatty) rats. Exercise and low body weight play a significant role in reducing calpains expression or elevating the calpains degradation in the skeletal muscle of NIDDM rats. Numerous investigations have been reported that non-coding polymorphisms in CAPN10 proteins might be involved in the NIDDM. Calpain and its mRNA presence had been reported in tissues from many mammalian species. CAPN10 and other calpains seem to be linked to glucose metabolism, insulin secretion and action pathways. This review will give an overview of the role of calpain in NIDDM and IDDM.     

Calpain and calpastatin

Calpain is a Ca2(+)-dependent cysteine endopeptidase and calpastatin is a calpain-specific endogenous inhibitor protein. Both calpain and calpastatin are very widely distributed in various animal tissues and cells. Low (microM) Ca2(+)-requiring calpain I and high (mM) Ca2(+)-requiring calpain II are known to exist. Calpain consists of one heavy (80 kDa) and one light (30 kDa) subunit. The heavy subunits of calpains I and II are different genetic products, whereas the light subunits are the same for both calpains I and II. Molecular cloning as well as protein sequencing revealed that the heavy subunit has four domains, while the light subunit has two domains. The carboxyl terminal domain of each subunit is a calmodulin-like domain, whereas the catalytic site is located in domain 2 of the heavy subunit. Calpastatin has four internally repetitive inhibitory domains. A single domain, or even a truncated 27-mer fragment thereof, possesses inhibitory activity against calpains. Calpain shows a rather broad substrate specificity. It can cleave various enzymes, and cytoskeletal, membrane and receptor proteins. Calpain-catalyzed activation of protein kinase C and transglutaminase may represent a few of the physiological functions of calpain, but a great many other functions can be assigned as well to calpain. Immunohistochemical studies revealed very wide but quite diverse distribution of calpains I and II and calpastatin among various tissues and cells. The expression of the genes for calpain and calpastatin is found to be modulated by retrovirus (HTLV-I) infection to T-lymphocytes. The physiological significance of the calpain and calpastatin system is yet to be elucidated, and accumulating information definitely suggested the role of calpain/calpastatin in health and disease.     

A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity

Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43‐year‐old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild‐type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.     

Muscle‐specific calpain‐3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction

CAPN3 (also called p94/calpain‐3) is a skeletal muscle‐specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb‐girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril‐based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.     

Modification of SARS-CoV S1 gene render expression in Pichia pastoris

S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777–1683, n.t.1041–1050, n.t.1236–1248, n.t.1317–1335, n.t.1590–1605; based on the same amino acid sequences. The modified gene was cloned into a yeast expression vector pPIC9K. The resultant plasmid pPIC9K- S1 was transformed into Pichia pastoris GS 115 and the protein expression was induced with methanol. SDS-PAGE confirmed that the recombinant SI was secreted in the supernatant of induced GS 115. The protein yield reached 69 mg/l. ELISA and Western blot demonstrated that the S1 could react with the convalescent sera of people infected by SARS-CoV. Furthermore, ligand blot assay showed that the recombinant S1 could react with ACE2, the receptor of SARS-CoV. The molecular mass of expressed S1 was about 70 kDa, which was higher than that of the 30 kDa expected. PNGase F deglycosylation resulted in a protein band of 30 kDa. In conclusion, the S1 gene modification rendered the high-level expression of S1 in P. pastoris GS 115 and the protein was secreted as a biologically active form which was hyperglycosylated.     

The calpain system

The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.     

Induction of glucocorticoid receptor‐β expression in epithelial cells of asthmatic airways by T‐helper type 17 cytokines

Background Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor‐β (GR‐β) in many cell types. T‐helper type 17 (Th17) cytokine (IL‐17A and F) expressions increase in mild and in difficult‐to‐treat asthma. We hypothesize that IL‐17A and F cytokines alone or in combination, induce the expression of GR‐β in bronchial epithelial cells. Objectives To confirm the expression of the GR‐β and IL‐17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL‐17A and F on the expression of GR‐β in bronchial epithelial cells obtained from normal subjects and asthmatic patients. Methods The expression of IL‐17A and F, GR‐α and GR‐β was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q‐RT‐PCR. Immunohistochemistry for IL‐17 and GR‐β was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL‐6 in response to IL‐17A and F and dexamethasone was determined by Q‐RT‐PCR using primary airway epithelial cells from normal and asthmatic subjects. Results We detected significantly higher levels of IL‐17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR‐α expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL‐17F and GR‐β in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR‐β in response to IL‐17A and F in the cells from asthmatics (P0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL‐17‐induced IL‐6 expression in cells from normal individuals but not in those from asthmatics (P0.05). Conclusion Evidence of an increased GR‐β expression in epithelial cells following IL‐17 stimulation suggests a possible role for Th17‐associated cytokines in the mechanism of steroid hypo‐responsiveness in asthmatic subjects. Cite this as: A. Vazquez‐Tello, A. Semlali, J. Chakir, J. G. Martin, D. Y. Leung, D. H. Eidelman and Q. Hamid, Clinical & Experimental Allergy, 2010 (40) 1312–1322.     

Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model

Activation of the innate immune system is critical for clearance of bacterial pathogens to limit systemic infections and host tissue damage. Here, we report a key role for calpain proteases in bacterial clearance in mice with acute peritonitis. Using transgenic mice expressing Cre recombinase primarily in innate immune cells (fes‐Cre), we generated conditional capns1 knockout mice. Consistent with capns1 being essential for stability and function of the ubiquitous calpains (calpain‐1, calpain‐2), peritoneal cells from these mice had reduced levels of calpain‐2/capns1, and reduced proteolysis of their substrate selenoprotein K. Using an acute bacterial peritonitis model, we observed impaired bacterial killing within the peritoneum and development of bacteremia in calpain knockout mice. These defects correlated with significant reductions in IL‐1α release, neutrophil recruitment, and generation of reactive oxygen species in calpain knockout mice with acute bacterial peritonitis. Peritoneal macrophages from calpain knockout mice infected with enterobacteria ex vivo, were competent in phagocytosis of bacteria, but showed impaired clearance of intracellular bacteria compared with control macrophages. Together, these results implicate calpains as key mediators of effective innate immune responses to acute bacterial infections, to prevent systemic dissemination of bacteria that can lead to sepsis.     

Antigen 43/Fcε3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine

The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and β subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti‐asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self‐molecules.     

Casein kinase 2‐dependent phosphorylation of human Rad9 mediates the interaction between human Rad9‐Hus1‐Rad1 complex and TopBP1

The checkpoint clamp Rad9‐Hus1‐Rad1 (9‐1‐1) is loaded by the Rad17–RFC complex onto chromatin after DNA damage and plays a key role in the ATR‐dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9‐1‐1 and phosphorylates serines 341 and 387 (Ser‐341 and Ser‐387) in the C‐terminal tail of Rad9. Interestingly, phosphorylated Ser‐387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9‐1‐1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over‐expressed in HeLa cells, a mutant Rad9 harboring phospho‐deficient substitutions at both Ser‐341 and Ser‐387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR‐dependent checkpoint pathway through its ability to phosphorylate Ser‐341 and Ser‐387 of the Rad9 subunit of the 9‐1‐1 complex.     

Reporter gene construct containing 1.4‐kB α1‐proteinase inhibitor promoter confers expression in the cornea of transgenic mice

A 1.4‐kb human α1‐proteinase inhibitor (α1‐PI) 5′‐flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The 1.4‐kb α1‐PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain. This finding implies that the α1‐PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments. Anat Rec 266:5–9, 2002. © 2002 Wiley‐Liss, Inc.     

Molecular Cloning,Expression, Purification and Immunological Characterization of Three Low‐Molecular Weight Proteins Encoded by Genes in Genomic Regions of Difference of Mycobacterium Tuberculosis

The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene‐specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM‐T Easy and subcloned into pGES‐TH‐1 vector for high‐level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione‐S‐transferase (GST)‐Rv3874, GST‐Rv3875 and GST‐Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione‐Sepharose and Ni‐NTA agarose affinity matrix after cleavage of the column‐bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen‐specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen‐specific antibodies in rabbits and demonstrate the usefulness of pGES‐TH‐1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.     

Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended‐spectrum β‐lactamases

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended‐spectrum β‐lactamase (ESBL)‐positive Klebsiella pneumoniae producing CTX‐M‐15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, β‐lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital‐associated materials was analysed. CTX‐M‐15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY‐2, DHA‐1). Independent of plasmid‐mediated beta‐lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC‐producing isolates. In conclusion, ESBL‐producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL‐producing E. coli strains.     

The Role of Epistasis in the Etiology of Polycystic Ovary Syndrome among Indian Women: SNP‐SNP and SNP‐Environment Interactions

Polycystic Ovary Syndrome (PCOS) is the most common endocrinopathy in women of reproductive age. It is a heterogeneous androgen excess disorder determined by the interaction of multiple genetic and environmental factors. Our earlier analysis on a panel of six candidate genes (Androgen receptor CAG repeats, Follistatin, Luteinizing hormone β subunit, Calpain10, Insulin receptor substrate‐1 and PPARγ) based on 250 PCOS cases and 299 controls revealed significant association patterns with PCOS among South‐Indian women. We report here for the first time, the SNP‐SNP and SNP‐environment interactions of these genes in the same cohort. Both multivariate logistic regression as well as epistasis analysis (using Multifactor dimensionality reduction software) yielded significant results (P < 0.05). All CAPN10 SNPs show association (either risk‐conferring or protective) in the obese group, highlighting the importance of this gene in the PCOS pathophysiology. LHP7(LHβ) and UCSNP44(CAPN10) emerged to be the prominent SNPs in the SNP‐SNP interaction analysis. The best SNP‐SNP interaction model was obtained between CAPN10 UCSNP‐44 and PPARγ His447His, implying a significant metabolic component in the PCOS pathology. Replicating our findings in BMI‐specific cohorts in different ethnic populations would be warranted in future to identify the physiological networks in PCOS.