Tandem Mass Tag-Labeled Quantitative Proteome Analyses Identify C1R and A2M as Novel Serum Biomarkers in Pregnant Women with Obstructive Sleep Apnea.

The aim of this study was to identify serum diagnostic biomarkers associated with the severity of obstructive sleep apnea (OSA) during pregnancy. Differentially expressed proteins (DEPs) were identified in the control (C), mild (O), and moderate (MO) OSA groups (n = 3 in each group). Bioinformatics analysis was conducted to identify the underlying functions, pathways, and networks of the proteins. Receiver operating characteristic curves were used to assess the diagnostic value of the identified DEPs. The enzyme-linked immunoassay was performed to detect serum levels of the complement C1r subcomponent (C1R) and alpha-2-macroglobulin (A2M) in 79 pregnant women with OSA (mild OSA [n = 32]; moderate OSA [n = 29], and severe OSA [n = 18]) and 65 healthy pregnant women without OSA. Pearson's correlation analysis was conducted to analyze the correlation between C1R and A2M levels and OSA clinicopathological factors. In total, 141 DEPs, 29 DEPs, and 103 DEPs were identified in the three groups (i.e., the mild OSA vs control group, the moderate OSA vs mild apnea group, and the moderate OSA vs control group, respectively). C1R and A2M were identified as continuously up-regulated proteins, and the levels of C1R and A2M were associated with OSA severity. C1R and A2M were found to be correlated with body mass index, systolic blood pressure, apnea-hypopnea index, oxygen desaturation index, time with saturation below 90%, and lowest SaO2. Adverse maternal and neonatal outcomes were observed in pregnant women with OSA. C1R and A2M have been identified as diagnostic biomarkers and are associated with the severity of OSA during pregnancy.

Conformational dynamics of complement protease C1r inhibitor proteins from Lyme disease- and relapsing fever-causing spirochetes.

Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever-causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors.

Downregulation of C1R promotes hepatocellular carcinoma development by activating HIF-1α-regulated glycolysis.

C1R has been identified to have a distinct function in cutaneous squamous cell carcinoma that goes beyond its role in the complement system. However, it is currently unknown whether C1R is involved in the progression of hepatocellular carcinoma (HCC). HCC tissues were used to examine C1R expression in relation to clinical and pathological factors. Malignant characteristics of HCC cells were assessed through in vitro and in vivo experiments. The mechanism underlying the role of C1R in HCC was explored through RNA-seq, methylation-specific PCR, immuno-precipitation, and dual-luciferase reporter assays. This study found that the expression of C1R decreased as the malignancy of HCC increased and was associated with poor prognosis. C1R promoter was highly methylated through DNMT1 and DNMT3a, resulting in a decrease in C1R expression. Downregulation of C1R expression resulted in heightened malignant characteristics of HCC cells through the activation of HIF-1α-mediated glycolysis. Additionally, decreased C1R expression was found to promote xenograft tumor formation. We found that C-reactive protein (CRP) binds to C1R, and the free CRP activates the NF-κB signaling pathway, which in turn boosts the expression of HIF-1α. This increase in HIF-1α leads to higher glycolysis levels, ultimately promoting aggressive behavior in HCC. Methylation of the C1R promoter region results in the downregulation of C1R expression in HCC. C1R inhibits aggressive behavior in HCC in vitro and in vivo by inhibiting HIF-1α-regulated glycolysis. These findings indicate that C1R acts as a tumor suppressor gene during HCC progression, opening up new possibilities for innovative therapeutic approaches.

Oral characteristics in adult individuals with periodontal Ehlers-Danlos syndrome.

AIM: Periodontal Ehlers-Danlos syndrome (pEDS) is a monogenic type of Ehlers-Danlos syndrome characterized by periodontal destruction at a young age. The present study aimed to document the oral phenotype of pEDS based on prospective clinical investigations. MATERIALS AND METHODS: Thirty-five adult individuals from 13 families with a clinically and genetically confirmed diagnosis of pEDS underwent a systematic oral assessment. RESULTS: Periodontitis stage 3 or 4 or edentulism due to periodontal destruction were diagnosed in 94% of the individuals. First permanent tooth loss was reported at the age of 21.5 years (median; range 13-43 years). Deep periodontal pockets were infrequent, with 94% measuring <4 mm. However, there was increased clinical attachment loss (CAL) averaging 8 mm (range 4-13 mm), and the probability of being edentate between the age of 35 and 44 years was 28-47% compared with less than 0.25% of the general population. Radiographic anomalous findings were only found in a portion of subjects and consisted of fused roots of maxillary second molars (81%), root hypoplasia (57%), taurodontism (26%) and tooth rotation of premolars (67%). As such, radiographic findings are not considered common characteristics of pEDS. CONCLUSIONS: Characteristic oral traits of pEDS in adults are severe CAL with shallow probing depths and marked gingival recession. This is complemented by a lack of attached gingiva. These indications need to be paralleled by genetic analyses to diagnose pEDS unambiguously.

HLA class II immunopeptidomics reveals that co-inherited HLA-allotypes within an extended haplotype can improve proteome coverage for immunosurveillance.

Human leucocyte antigen (HLA) class II molecules in humans are encoded by three different loci, HLA-DR, -DQ, and -DP. These molecules share approximately 70% sequence similarity and all present peptide ligands to circulating T cells. While the peptide repertoires of numerous HLA-DR, -DQ, and -DP allotypes have been examined, there have been few reports on the combined repertoire of these co-inherited molecules expressed in a single cell as an extended HLA haplotype. Here we describe the endogenous peptide repertoire of a human B lymphoblastoid cell line (C1R) expressing the class II haplotype HLA-DR12/DQ7/DP4. We have identified 71350 unique naturally processed peptides presented collectively by HLA-DR12, HLA-DQ7, or HLA-DP4. The resulting "haplodome" is complemented by the cellular proteome defined by standard LC-MS/MS approaches. This large dataset has shed light on properties of these class II ligands especially the preference for membrane and extracellular source proteins. Our data also provides insights into the co-evolution of these conserved haplotypes of closely linked and co-inherited HLA molecules; which together increase sequence coverage of cellular proteins for immune surveillance with minimal overlap between each co-inherited HLA-class II allomorph.

Prospective clinical investigations of children with periodontal Ehlers-Danlos syndrome identify generalized lack of attached gingiva as a pathognomonic feature.

PURPOSE: We report prospective clinical investigations of children affected with periodontal Ehlers-Danlos syndrome (pEDS). The main clinical features of pEDS in adults are early severe periodontitis, generalized lack of attached gingiva, and pretibial hemosiderin plaques due to dominant pathogenic variants in the C1R or C1S genes. METHODS: Nineteen children with a parent diagnosed with molecularly confirmed pEDS underwent physical examination including oral and radiological investigations followed by genetic testing. RESULTS: The only consistent manifestation of pEDS in childhood was a characteristic gingival phenotype: generalized lack of attached gingiva. All children with this gingival phenotype had inherited the familial pathogenic variant (n = 12) whereas the gingival phenotype was absent in children without the familial pathogenic variant (n = 7). Easy bruising was reported in eight affected and zero unaffected children. Other manifestations of pEDS were rarely present in children. Only 2/12 affected children aged 8 and 13 years fulfilled the clinical criteria for pEDS. CONCLUSION: Generalized lack of attached gingiva is a pathognomonic feature of pEDS and the only clinical finding that is consistently present in affected adults and children. This is important because an early diagnosis may facilitate better dental hygiene in childhood, which may be essential to prevent early dental loss.

Weighted burden analysis of exome-sequenced late-onset Alzheimer's cases and controls provides further evidence for a role for PSEN1 and suggests involvement of the PI3K/Akt/GSK-3ß and WNT signalling pathways.

Previous studies have implicated common and rare genetic variants as risk factors for late-onset Alzheimer's disease (LOAD). Here, weighted burden analysis was applied to over 10,000 exome-sequenced subjects from the Alzheimer's Disease Sequencing Project. Analyses were carried out to investigate whether rare variants predicted to have a functional effect within a gene were more commonly seen in cases or in controls. Confirmatory results were obtained for TREM2, ABCA7, and SORL1. Additional support was provided for PSEN1 (p = 0.0002), which previously had been only weakly implicated in LOAD. There was suggestive evidence that functional variants in PIK3R1, WNT7A, C1R, and EXOC5 might increase risk and that variants in TIAF1 and/or NDRG2 might have a protective effect. Overall, there was strong evidence (p = 5 × 10-6 ) that variants in tyrosine phosphatase genes reduce the risk of developing LOAD. Because PIK3R1 variants are expected to impair PI3K/Akt/GSK-3ß signalling while variants in tyrosine phosphatase genes would enhance it, these findings are in line with those from animal models, suggesting that this pathway is protective against Alzheimer's disease.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Complement C1r serine protease contributes to kidney fibrosis.

We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-ß), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.

A Chinese family with periodontal Ehlers-Danlos syndrome associated with missense mutation in the C1R gene.

AIM: We report a rare case of the periodontal Ehlers-Danlos syndrome (pEDS) associated with severe periodontitis and tooth loss in a Chinese family. MATERIAL AND METHODS: The proband complained of gingival bleeding and mobility, which were also reported in his mother, and maternal uncle and his maternal grandmother and great-grandfather in the past. All family members underwent oral, physical, dermatological, and genetic examinations. RESULTS: Oral manifestation of family members affected with pEDS presented severe periodontitis with multiple or total tooth loss. The proband displayed unique clinical manifestations including a characteristic facial appearance and thin, translucent skin with easily visible venous patterns on feet. Whole-exome sequencing identified missense mutation c.265T>C in C1R in all affected family members tested and frameshift mutation c.1322delG in COL3A1 in the proband alone. None of the unaffected members showed any marked oral, physical, dermatological, or genetic findings. CONCLUSION: We reported an extremely rare case of pEDS with a missense mutation in C1R in a Chinese family, with coexistence C1R and COL3A1 mutations in the proband who was suspected to suffer from vascular EDS as well. To our knowledge, this is the first case of coexistence of two forms of EDS in a single individual.

C1r and C1s from Nile tilapia (Oreochromis niloticus): Molecular characterization, transcriptional profiling upon bacterial and IFN-γ inductions and potential role in response to bacterial infection.

The complement components C1r and C1s play a vital role in immunity with the activation of C1 complex in the classical complement pathway against pathogen infection. In this study, Nile tilapia (Oreochromis niloticus) C1r and C1s orthologs (OnC1r and OnC1s) were identified and characterized. The cDNA of OnC1r and OnC1s ORFs consisted of 1902 bp and 2100 bp of nucleotide sequence encoding polypeptides of 633 and 699 amino acids, respectively. The deduced OnC1r and OnC1s proteins both possessed CUB, EGF, CCP and SP domains, which were significantly homology to teleost. Spatial mRNA expression analysis revealed that the OnC1r and OnC1s were highly expressed in liver. After the in vivo challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), the mRNA expressions of OnC1r and OnC1s were significantly up-regulated in liver and spleen, which were consistent with immunohistochemical detection at the protein level. The up-regulation of OnC1r and OnC1s expressions were also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, S. agalactiae, and recombinant OnIFN-γ. Taken together, the results of this study indicated that OnC1r and OnC1s were likely to get involved in the immune response of Nile tilapia against bacterial infection.

Molecular determinants of the substrate specificity of the complement-initiating protease, C1r.

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

Mechanism of an unusual, but clinically significant, digoxin-bupropion drug interaction.

An unusual, but clinically significant, digoxin (DIG)-bupropion (BUP) drug interaction (DDI), in which BUP increased DIG renal clearance by 80% is reported. To investigate the mechanism(s) of this unusual DDI, first the effect of BUP, its circulating metabolites or their combination on [(3) H]-DIG transport by cells expressing human P-gp or human OATP4C1 was determined. Second, the study asked whether this DDI could be replicated in the rat so that it could be used to conduct mechanistic studies. Then, the effect of BUP and its rat metabolites on [(3) H]-DIG transport were tested by cells expressing rat Oatp4c1. Bupropion and its metabolites had no effect on human P-gp mediated transepithelial transport of [(3) H]-DIG. Bupropion and hydroxybupropion (HBUP) significantly stimulated H-OATP4C1 mediated transport of [(3) H]-DIG. In addition, BUP cocktail (BUP plus its metabolites) significantly increased the H-OATP4C1 mediated transport of [(3) H]-DIG, and partially reversed the inhibition by 100 µm DIG. However, erythro-hydrobupropion (EBUP) and threo-hydrobupropion (TBUP) did not affect the [(3) H]-DIG uptake by H-OATP4C1 cells. Bupropion administration significantly increased digoxin renal clearance in rats. Surprisingly, bupropion significantly inhibited r-Oatp4c1 mediated transport of [(3) H]-DIG at clinically relevant unbound plasma concentrations of BUP or those observed in the rat study, while HBUP or TBUP did not. These data support our hypothesis that at clinically relevant plasma concentrations, bupropion and its metabolites activate H-OATP4C1 mediated DIG tubular secretion, and could possibly explain the increase in digoxin renal clearance produced by bupropion. While bupropion increased digoxin renal clearance in the rat, it appeared to do so by inhibiting r-Oatp4c1-mediated digoxin renal reabsorption.

Tumor Cell-Derived Complement Component C1r Acts as a Prognostic Biomarker and Promotes Esophageal Squamous Cell Carcinoma Progression.

BACKGROUND: Mounting evidence indicates that complement components play a crucial role in cancer progression. Recent findings indicate that certain complement components display a significant rise in expression within esophageal squamous cell carcinoma (ESCC). However, the specific tumorigenic functions of these components remain unclear. This study focuses on investigating the expression pattern of C1r, elucidating a role for C1r in ESCC, as well as exploring underlying mechanisms controlled by C1r. METHODS: The expression of C1r in ESCC tissues, malignant epithelial cells, and its relationship with survival were analyzed using the Gene Expression Omnibus (GEO) database and tissue microarrays. Single-cell RNA sequencing (scRNA-seq) was used to study the expression of C1r in malignant epithelial cells. C1r knockdown or C1r overexpression in cultured ESCC cells were used to assess the effects of C1r on proliferation, migration, invasion, cell-matrix adhesion, apoptosis, and growth of xenografted tumors in immunocompromised (nude) mice. Western blotting was used to detect the expression of MMP-1 and MMP-10 in C1r knockdown or C1r overexpressing ESCC cells. RESULTS: C1r was highly expressed in ESCC tissues, malignant epithelial cells, and cultured ESCC cell lines. High C1r expression indicated a poor prognosis. Knockdown of C1r significantly suppressed the proliferation, migration, invasion, cell-matrix adhesion, and promoted apoptosis in cultured ESCC cells. Additionally, knockdown of C1r markedly inhibited tumor growth in nude mice. Overexpression of C1r had the opposite effects. C1r induced the expression of MMP-1 and MMP-10. CONCLUSIONS: C1r is highly expressed in ESCC and promotes the progression of this tumor type. Our findings suggest that C1r may serve as a novel prognostic biomarker and therapeutic target in ESCC.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs.

Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome.

Degradation of collagen I by activated C1s in periodontal Ehlers-Danlos Syndrome.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.

The Relationship between Complement Components C1R and C5 Gene Polymorphism and the Values of Blood Indices in Suckling Piglets.

The main mechanism of innate immunity is the complement system. Its components include the protein products of the C1R and C5 genes, which are involved in the classical activation pathway as well as the inflammatory and cytolytic immune responses, respectively. The aim of this study was to determine the relationship between PCR-restriction fragment length polymorphism in C1R (726T > C) and C5 (1044A > C) genes, and the values of hematological and biochemical blood indices in suckling crossbred (Polish Large White × Polish Landrace × Duroc × Pietrain) piglets (n = 473), considering their age (younger, 21 ± 3 days, n = 274; older, 35 ± 3 days, n = 199) and health status. The frequencies of the C5 genotypes deviated from the Hardy-Weinberg expectations. Younger piglets, healthy piglets, piglets that deviated from physiological norms and older piglets with the C1R TT genotype all had lower white and red blood cell indices. In piglets with the C5 CC genotype, younger piglets, piglets that deviated from physiological norms and older piglets, a greater number and/or percentage of monocytes were recorded in the blood. Older piglets also showed an increase in the number of leukocytes and granulocytes, along with a tendency for a decrease in the percentage of lymphocytes in their blood. We concluded that a polymorphism in the C1R gene may exhibit a functional association or genetic linkage with other genes involved in the process of erythropoiesis. Furthermore the relationship between the C5 gene polymorphism and the number and/or percentage of monocytes in the blood may modify the body's defense abilities. Piglets with the CC genotype, having an increased number/proportion of these cells in their blood, probably display a weakened immune response to pathogens or a chronic stimulation of the immune system.

A novel mechanism underlying allosteric regulation of ADAMTS-13 revealed by hydrogen-deuterium exchange plus mass spectrometry.

Background: ADAMTS-13, a plasma metalloprotease, cleaves von Willebrand factor. ADAMTS-13 activity appears to be regulated through allosteric inhibition by its distal C-terminus. Objectives: The objective of this study was to better understand how domain-domain interactions may affect ADAMTS-13 conformations and functions. Methods: We performed deuterium-hydrogen exchange plus mass spectrometry to assess the number and rate of deuterium incorporation into various peptides of full-length ADAMTS-13 and its truncated variants. Results: Under physiological conditions, a bimodal distribution of deuterium incorporation was detected in the peptides from metalloprotease (217-230 and 282-304), cysteine-rich (446-482), and CUB (for complement C1r/C1s, Uegf, Bmp1) domains (1185-1214, 1313-1330, 1341-1347, 1358-1378, and 1393-1407) of full-length recombinant ADAMTS-13, but not of truncated variants. These results suggest that the full-length ADAMTS-13 undergoes conformational changes. On removal of the middle and distal C-terminal domains, the number and rate of deuterium incorporation were increased in the peptides from cysteine-rich (445-467, 467-482, and 495-503) and spacer domains (621-642 and 655-654) but decreased in the peptides from metalloprotease (115-124, 217-230, and 274-281). Moreover, most peptides, except for 217-230 and 1357-1376, exhibited a pD-dependent deuterium incorporation in the full-length ADAMTS-13, but not in the truncated variant (eg, MDTCS or T5C). These results further suggest that the bimodal deuterium incorporation observed in the peptides from the full-length ADAMTS-13 is the result of potential impact from the middle to distal C-terminal domains. Surface plasmon resonance revealed the direct binding interactions between the distal and proximal domains of ADAMTS-13. Conclusion: Our results provide novel insight on how intramolecular interactions may affect conformations of ADAMTS-13, thus regulating its proteolytic functions.