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1.
J Immunol ; 212(4): 645-662, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38180157

RESUMO

Collectin is a crucial component of the innate immune system and plays a vital role in the initial line of defense against pathogen infection. In mammals, collectin kidney 1 (CL-K1) is a soluble collectin that has recently been identified to have significant functions in host defense. However, the evolutionary origins of immune defense of CL-K1 and its mechanism in clearance of pathogenic microorganisms remain unclear, especially in early vertebrates. In this study, the Oreochromis niloticus CL-K1 (OnCL-K1) protein was purified and identified, which was capable of binding to two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila. Interestingly, OnCL-K1 exhibited direct bactericidal activity by binding to lipoteichoic acid or LPS on cell walls, disrupting the permeability and integrity of the bacterial membrane in vitro. Upon bacterial challenge, OnCL-K1 significantly inhibited the proliferation of pathogenic bacteria, reduced the inflammatory response, and improved the survival of tilapia. Further research revealed that OnCL-K1 could associate with OnMASPs to initiate and regulate the lectin complement pathway. Additionally, OnCD93 reduced the complement-mediated hemolysis by competing with OnMASPs for binding to OnCL-K1. More importantly, OnCL-K1 could facilitate phagocytosis by collaborating with cell surface CD93 in a lectin pathway-independent manner. Moreover, OnCL-K1 also promoted the formation of phagolysosomes, which degraded and killed ingested bacteria. Therefore, this study reveals the antibacterial response mechanism of CL-K1 in primitive vertebrates, including promoting complement activation, enhancing opsonophagocytosis, and killing of macrophages, as well as its internal links, all of which provide (to our knowledge) new insights into the understanding of the evolutionary origins and regulatory roles of the collectins in innate immunity.


Assuntos
Macrófagos , Opsonização , Animais , Macrófagos/metabolismo , Ativação do Complemento , Rim/metabolismo , Vertebrados , Colectinas/metabolismo , Proteínas de Peixes/metabolismo , Mamíferos/metabolismo
2.
Cell Death Dis ; 14(11): 785, 2023 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-38036508

RESUMO

Hepatic stellate cell is one of the major nonparenchymal cell types in liver. It has been proved the hepatic stellate cells are activated upon liver injury and produce excessive extracellular matrix to induce liver fibrosis. Single-cell RNA sequencing has been introduced to identify the subpopulations and function of hepatic stellate cells for its remarkable resolution of representation of single-cell transcriptome. According to the re-analysis of single-cell RNA sequencing data and pseudotime trajectory inference, we have found the C-type lectins including Colec10 and Colec11 are not produced by hepatocytes but predominantly produced by hepatic stellate cells, especially quiescent ones in the mice livers. In addition, the expression of Colec10 is decreased in the fibrotic livers of CCl4-challenged mice. COLEC10 is also mainly expressed in the hepatic stellate cells of human livers and the expression of COLEC10 is decreased with the progression of liver fibrosis. The bulk RNA sequencing data of the lentivirus transfected LX-2 cells indicates the function of COLEC10 is associated with inflammation, angiogenesis and extracellular matrix alteration. Surprisingly, the in vitro overexpression of COLEC10 in LX-2 cells promotes the mRNA expression of extracellular matrix components including COL1A1, COL1A2 and COL3A1 and the extracellular matrix degradation enzyme MMP2. To further investigate the role of COLEC10 in the pathogenesis of liver fibrosis, the serum concentration of COLEC10 in patients with chronic liver disease and healthy donors is measured. The serum concentration of COLEC10 is elevated in the patients with chronic liver disease compared to the healthy donors and positively correlated with serum concentration of the D-dimer but not the most of liver function markers. Altogether, we conclude that the C-type lectin COLEC10 is predominantly produced by the hepatic stellate cells and involved in the pathogenesis of liver fibrosis.


Assuntos
Células Estreladas do Fígado , Hepatopatias , Humanos , Camundongos , Animais , Células Estreladas do Fígado/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Cirrose Hepática/patologia , Fígado/metabolismo , Hepatopatias/metabolismo , Colectinas/metabolismo
3.
Cells ; 12(13)2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37443840

RESUMO

Retinal pigment epithelium (RPE) cell allotransplantation is seen as a possible solution to retinal diseases. However, the RPE-complement system triggered by the binding of collectin-11 (CL-11) is a potential barrier for RPE transplantation as the complement-mediated inflammatory response may promote T cell recognition. To address this, we investigated the role of CL-11 on T cell immuno-response. We confirmed that RPE cells up-regulated MHC class I and expressed MHC class II molecules in an inflammatory setting. Co-cultures of RPE cells with T cells led to the inhibition of T cell proliferation. We found that CL-11 was partially responsible for this effect as T cell binding of CL-11 inhibited T cell proliferation in association with the downregulation of CD28. We also found that the suppressive action of CL-11 was abrogated in the presence of the RGD peptide given to block the T cell binding of CL-11 by its collagen-like domain. Because RPE cells can bind and secrete CL-11 under stress conditions, we postulate that soluble CL-11 contributes to the immunosuppressive properties of RPE cells. The investigation of this dual biological activity of CL-11, namely as a trigger of the complement cascade and a modulator of T cell responses, may provide additional clues about the mechanisms that orchestrate the immunogenic properties of RPE cells.


Assuntos
Epitélio Pigmentado da Retina , Linfócitos T , Linfócitos T/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Células Cultivadas , Células-Tronco/metabolismo , Colectinas/metabolismo , Células Epiteliais/metabolismo
4.
JCI Insight ; 8(5)2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36883567

RESUMO

Collectin-11 (CL-11) is a recently described soluble C-type lectin that has distinct roles in embryonic development, host defence, autoimmunity, and fibrosis. Here we report that CL-11 also plays an important role in cancer cell proliferation and tumor growth. Melanoma growth was found to be suppressed in Colec11-/- mice in a s.c. B16 melanoma model. Cellular and molecular analyses revealed that CL-11 is essential for melanoma cell proliferation, angiogenesis, establishment of more immunosuppressive tumor microenvironment, and the reprogramming of macrophages to M2 phenotype within melanomas. In vitro analysis revealed that CL-11 can activate tyrosine kinase receptors (EGFR, HER3) and ERK, JNK, and AKT signaling pathways and has a direct stimulatory effect on murine melanoma cell proliferation. Furthermore, blockade of CL-11 (treatment with L-fucose) inhibited melanoma growth in mice. Analysis of open data sets revealed that COLEC11 gene expression is upregulated in human melanomas and that high COLEC11 expression has a trend toward poor survival. CL-11 also had direct stimulatory effects on human tumor cell proliferation in melanoma and several other types of cancer cells in vitro. Overall, our findings provide the first evidence to our knowledge that CL-11 is a key tumor growth-promoting protein and a promising therapeutic target in tumor growth.


Assuntos
Proliferação de Células , Colectinas , Melanoma Experimental , Neoplasias Cutâneas , Animais , Humanos , Camundongos , Autoimunidade , Proliferação de Células/genética , Proliferação de Células/fisiologia , Colectinas/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Proteínas de Neoplasias , Receptores Proteína Tirosina Quinases , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia
5.
Genet Med ; 24(8): 1653-1663, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35511137

RESUMO

PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.


Assuntos
COVID-19 , Idoso , COVID-19/genética , Colectinas/genética , Colectinas/metabolismo , Células Germinativas , Humanos , Lectinas/genética , SARS-CoV-2 , Sequenciamento do Exoma
6.
BMC Cancer ; 22(1): 380, 2022 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-35397600

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is the third-most deadly cancer worldwide. More breakthroughs are needed in the clinical practice for liver cancer are needed, and new treatment strategies are required. This study aims to determine the significant differences in genes associated with LIHC and further analyze its prognostic value further. METHODS: Here, we used the TCGA-LIHC database and the profiles of GSE25097 from GEO to explore the differentially co-expressed genes in HCC tissues compared with paratumor (or healthy) tissues. Then, we utilized WGCNA to screen differentially co-expressed genes. Finally, we explored the function of FYN in HCC cells and xenograft tumor models. RESULTS: We identified ten hub genes in the protein-protein interaction (PPI) network, but only three (COLEC10, TGFBR3, and FYN) appeared closely related to the prognosis. The expression of FYN was positively correlated with the prognosis of HCC patients. The xenograft model showed that overexpression of FYN could significantly inhibit malignant tumor behaviors and promote tumor cell apoptosis. CONCLUSION: Thus, FYN may be central to the development of LIHC and maybe a novel biomarker for clinical diagnosis and treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-fyn , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Colectinas/genética , Colectinas/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-fyn/genética , Proto-Oncogenes
7.
Mol Genet Genomic Med ; 9(11): e1834, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34636477

RESUMO

BACKGROUND: 3MC syndrome type 3 is an autosomal recessive disorder caused by mutations in the COLEC10 gene besides other genes like COLEC11 and MASP1. This disorder is characterized by facial dysmorphism, cleft lip and palate, postnatal growth deficiency, cognitive impairment, hearing loss, craniosynostosis, radioulnar synostosis, genital and vesicorenal anomalies, cardiac anomalies, caudal appendage, and umbilical hernia. METHODS: In the present study, whole-exome sequencing was performed in order to identify disease causing variant in an Iranian 7-year-old affected girl with craniosynostosis, dolichocephaly, blepharoptosis, clinodactyly of the 5th finger, high myopia, long face, micrognathia, patent ductus arteriosus, downslanted palpebral fissures, telecanthus, and epicanthus inversus. Identified variant confirmation in the patient and segregation analysis in her family were performed using Sanger sequencing method. RESULTS: A novel homozygous frameshift deletion variant [NM_006438.5: c.128_129delCA; p.(Thr43AsnfsTer9)] was identified within the COLEC10 gene. Up to now, only three 3MC syndrome patients with mutations in the COLEC10 gene have been reported, and here, we report the fourth patient and the first homozygous frameshift variant. CONCLUSION: Other genes and factors responsible for 3MC syndrome occurrence are remained to be discovered. We believe further investigation of the genes in the lectin complement pathway is needed to be done for the identification of other causes of this disease.


Assuntos
Fenda Labial , Fissura Palatina , Criança , Fenda Labial/genética , Fissura Palatina/genética , Colectinas/genética , Colectinas/metabolismo , Feminino , Humanos , Irã (Geográfico) , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Sequenciamento do Exoma
8.
Front Immunol ; 12: 594858, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790889

RESUMO

The skin is a complex organ that faces the external environment and participates in the innate immune system. Skin immune homeostasis is necessary to defend against external microorganisms and to recover from stress to the skin. This homeostasis depends on interactions among a variety of cells, cytokines, and the complement system. Collectins belong to the lectin pathway of the complement system, and have various roles in innate immune responses. Mannose-binding lectin (MBL), collectin kidney 1, and liver (CL-K1, CL-L1) activate the lectin pathway, while all have multiple functions, including recognition of pathogens, opsonization of phagocytosis, and modulation of cytokine-mediated inflammatory responses. Certain collectins are localized in the skin, and their expressions change during skin diseases. In this review, we summarize important advances in our understanding of how MBL, surfactant proteins A and D, CL-L1, and CL-K1 function in skin immune homeostasis. Based on the potential roles of collectins in skin diseases, we suggest therapeutic strategies for skin diseases through the targeting of collectins and relevant regulators.


Assuntos
Colectinas/metabolismo , Homeostase , Imunidade Inata , Pele/imunologia , Pele/metabolismo , Animais , Biomarcadores , Colectinas/genética , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças , Humanos
9.
Dev Comp Immunol ; 122: 104108, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33909995

RESUMO

BLAST searches against databases for the bullfrog (Rana catesbeiana), using the collectin sequence previously identified in tadpoles, revealed the presence of at least 20 members of the collectin gene family. Phylogenetic analysis demonstrated that the bullfrog possesses expanded gene subfamilies encoding mannose-binding lectin (MBL) and pulmonary surfactant-associated protein D (PSAPD). Two collectins, of 20 kDa (PSAPD1) and 25 kDa (PSAPD6), were purified as a mixture from adult bullfrog plasma using affinity chromatography. These collectins were present as an oligomer of ~400 kDa in their native state, and showed Ca2+-dependent carbohydrate binding with different sugar preferences. Affinity-purified collectins showed weak E. coli agglutination and bactericidal activities, compared with those of plasma. Although both PSAPD1 and PSAPD6 genes were predominantly expressed in the liver, PSAPD1 transcripts were abundant in adults whereas PSAPD6 transcripts were abundant in tadpoles. The findings indicate that two gene subfamilies in the collectin family have diverged structurally, functionally and transcriptionally in the bullfrog. Rapid expansion of the collectin family in bullfrogs may reflect the onset of sub-functionalization of the prototype MBL gene towards tetrapod MBL and PSAPDs, and may be one means of natural adaptation in the innate immune system to various pathogens in both aquatic and terrestrial environments.


Assuntos
Carboidratos/imunologia , Imunidade Inata/imunologia , Lectina de Ligação a Manose/sangue , Proteína D Associada a Surfactante Pulmonar/sangue , Rana catesbeiana/metabolismo , Aglutinação/imunologia , Animais , Aderência Bacteriana/imunologia , Metabolismo dos Carboidratos/imunologia , Colectinas/sangue , Colectinas/genética , Colectinas/metabolismo , Escherichia coli/imunologia , Imunidade Inata/genética , Larva/imunologia , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Filogenia , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo
10.
Pharmacol Rev ; 73(2): 792-827, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33687995

RESUMO

The complement system was discovered at the end of the 19th century as a heat-labile plasma component that "complemented" the antibodies in killing microbes, hence the name "complement." Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies. SIGNIFICANCE STATEMENT: The complement system is the host's defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host's enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019.


Assuntos
COVID-19/epidemiologia , COVID-19/fisiopatologia , Proteínas do Sistema Complemento/fisiologia , Doenças Raras/fisiopatologia , Colectinas/metabolismo , Enzimas Ativadoras do Complemento/metabolismo , Complemento C3/metabolismo , Inativadores do Complemento/farmacologia , Terapia Genética/métodos , Humanos , Mediadores da Inflamação/metabolismo , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Pandemias , SARS-CoV-2 , Sinapses/metabolismo , Ficolinas
11.
Pediatr Nephrol ; 36(5): 1065-1073, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32472330

RESUMO

Ischaemia/reperfusion injury (IRI) is an inevitable and damaging consequence of the process of kidney transplantation, ultimately leading to delayed graft function and increased risk of graft loss. A key driver of this adverse reaction in kidneys is activation of the complement system, an important part of the innate immune system. This activation causes deposition of complement C3 on renal tubules as well as infiltration of immune cells and ultimately damage to the tubules resulting in reduced kidney function. Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement. CL-11 binds to a ligand that is exposed on the renal tubules by the stress caused by IRI, and through attached proteases, CL-11 activates complement and this contributes to the consequences outlined above. Recent work in our lab has shown that this damage-associated ligand contains a fucose residue that aids CL-11 binding and promotes complement activation. In this review, we will discuss the clinical context of renal transplantation, the relevance of the complement system in IRI, and outline the evidence for the role of CL-11 binding to a fucosylated ligand in IRI as well as its downstream effects. Finally, we will detail the simple but elegant theory that increasing the level of free fucose in the kidney acts as a decoy molecule, greatly reducing the clinical consequences of IRI mediated by CL-11.


Assuntos
Colectinas/metabolismo , Fucose/metabolismo , Transplante de Rim , Traumatismo por Reperfusão , Humanos , Rim , Transplante de Rim/efeitos adversos , Ligantes , Traumatismo por Reperfusão/etiologia
12.
Elife ; 92020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32909942

RESUMO

Properdin stabilizes the alternative C3 convertase (C3bBb), whereas its role as pattern-recognition molecule mediating complement activation is disputed for decades. Previously, we have found that soluble collectin-12 (sCL-12) synergizes complement alternative pathway (AP) activation. However, whether this observation is C3 dependent is unknown. By application of the C3-inhibitor Cp40, we found that properdin in normal human serum bound to Aspergillus fumigatus solely in a C3b-dependent manner. Cp40 also prevented properdin binding when properdin-depleted serum reconstituted with purified properdin was applied, in analogy with the findings achieved by C3-depleted serum. However, when opsonized with sCL-12, properdin bound in a C3-independent manner exclusively via its tetrameric structure and directed in situ C3bBb assembly. In conclusion, a prerequisite for properdin binding and in situ C3bBb assembly was the initial docking of sCL-12. This implies a new important function of properdin in host defense bridging pattern recognition and specific AP activation.


Assuntos
Colectinas , Via Alternativa do Complemento , Properdina , Aspergillus fumigatus/imunologia , Colectinas/sangue , Colectinas/metabolismo , Complemento C3/metabolismo , Via Alternativa do Complemento/imunologia , Via Alternativa do Complemento/fisiologia , Células HEK293 , Humanos , Properdina/análise , Properdina/metabolismo , Ligação Proteica/imunologia
13.
J Clin Lab Anal ; 34(11): e23469, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32822099

RESUMO

OBJECTIVE: To investigate the role of COLEC12 in osteosarcoma and observe the relationship between COLEC12 knockdown and the inflammation of osteosarcoma. Then, further explore whether the process is regulated by TLR4. METHOD: GEPIA and TCGA systems were used to predict the potential function of COLEC12. Western blot and RT-PCR were used to analyze the protein expression, or mRNA level, of COLEC12 in different tissue or cell lines. The occurrence and development of osteosarcoma were observed by using COLEC12 knockdown lentivirus. The inflammation indexes of osteosarcoma, in vitro and in vivo, were explored. TLR4 knockdown lentivirus was applied to the relationship between COLEC12 and TLR4. RESULTS: COLEC12 expression in SARC tumor tissue was higher than in normal, and a high expression of COLEC12 in SARC patients had a worse prognostic outcome. Pairwise gene correlation analysis revealed a potential relationship between COLEC12 and TLR4. The COLEC12 expression and mRNA level in the tumor or Saos-2 cells were increased. COLEC12 knockdown lentivirus could inhibit osteosarcoma development, in vivo and vitro, through reducing tumor volume and weight, weakening tumor proliferation, migration, and invasion, and enhancing apoptosis. Furthermore, COLEC12 knockdown could increase inflammation of osteosarcoma, in vivo and in vitro, through inducing myeloperoxidase (MPO), TLR4, NF-κB, and C3, and expression of related inflammatory factors. Finally, TLR4 knockdown lentivirus inhibits the progress of inflammation after COLEC12 regulation, in vivo and vitro. CONCLUSION: COLEC12 may be able to regulate apoptosis and inflammation of osteosarcoma, and TLR4 may be the downstream target factor of COLEC12 in inflammation.


Assuntos
Apoptose/genética , Colectinas , Inflamação/metabolismo , Osteossarcoma/metabolismo , Receptores Depuradores , Receptor 4 Toll-Like , Animais , Linhagem Celular Tumoral , Colectinas/genética , Colectinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
14.
Gene ; 761: 145036, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777525

RESUMO

Lupinus albus γ-conglutin is proposed to positively affect glucose metabolism through inhibition of hepatic glucose production and insulin-mimetic activity; however, the action mechanism is not entirely known. Besides, most studies had focused on its effect on molecular targets directly related to glucose metabolism, and few studies have investigated how γ-conglutin may affect the liver gene expression or if it plays a role in other metabolic processes. Therefore, we investigated the influence of γ-conglutin on the liver transcriptome of streptozotocin-induced diabetic rats using DNA microarrays, ontological analyses, and quantitative PCR. Of the 22,000 genes evaluated, 803 and 173 were downregulated and upregulated, respectively. The ontological analyses of the differentially expressed genes revealed that among others, the mitochondria, microtubules, cytoskeleton, and oxidoreductase activity terms were enriched, implying a possible role of γ-conglutin on autophagy. To corroborate the microarray results, we selected and quantified, by PCR, the expression of two genes associated with autophagy (Atg7 and Snx18) and found their expression augmented two and threefold, respectively; indicating a higher autophagy activity in animals treated with γ-conglutin. Although complementary studies are required, our findings indicate for the first time that the hypoglycaemic effects of γ-conglutin may involve an autophagy induction mechanism, a pivotal process for the preservation of cell physiology and glucose homeostasis.


Assuntos
Colectinas/farmacologia , Lupinus/metabolismo , Soroglobulinas/farmacologia , Transcriptoma/genética , Animais , Glicemia/metabolismo , Colectinas/metabolismo , Colectinas/fisiologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Fígado/patologia , Lupinus/genética , Masculino , Proteínas de Plantas/genética , Ratos , Ratos Wistar , Sementes/metabolismo , Soroglobulinas/metabolismo , Soroglobulinas/fisiologia
15.
J Immunol Methods ; 483: 112807, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32540393

RESUMO

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions may become eminent, with the result of high background signals. The workload associated with producing mAbs, able to withstand conjugation, preserving stability and affinity and avoiding off-target interactions, is comprehensive and related with only incidental success. We designed a method, where uncloned hybridomas were pre-selected for secretion of mAbs with the above characteristics. Using human collectin K1 (CL-K1, alias CL-11, Colec11) as a model antigen, mAbs present in culture supernatant from uncloned hybridomas were immobilized on Protein A beads, followed by solid phase biotinylation and subsequent elution. ELISA was employed to compare the binding activity of conjugated vs. unconjugated mAbs, and furthermore for their application in combination with other antibodies. From a group of 96 uncloned hybridomas we accomplished in obtaining five suitable mAbs, among which, two mAbs were superior. The successful conjugation of the selected mAbs with fluorophores and subsequent applications in microscopy and flow cytometry were further demonstrated. In conclusion, pre-selection of uncloned hybridomas, by testing of their mAbs' ability to withstand conjugation with tracers or drugs, is a successful strategy to avoid a huge workload of cloning numerous hybridomas, in order to obtain conjugatable mAbs.


Assuntos
Anticorpos Monoclonais/biossíntese , Colectinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunoconjugados/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Biotinilação , Células CHO , Clonagem Molecular , Colectinas/genética , Colectinas/imunologia , Cricetulus , Humanos , Hibridomas , Imunoconjugados/genética , Imunoconjugados/imunologia , Camundongos , Estabilidade Proteica , Proteína Estafilocócica A/imunologia
16.
J Biol Chem ; 295(27): 9157-9170, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32424040

RESUMO

C-type lectins that contain collagen-like domains are known as collectins. These proteins are present both in the circulation and in extravascular compartments and are central players of the innate immune system, contributing to first-line defenses against viral, bacterial, and fungal pathogens. The collectins mannose-binding lectin (MBL) and surfactant protein D (SP-D) are regulated by tissue fibroblasts at extravascular sites via an endocytic mechanism governed by urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180), which is also a collagen receptor. Here, we investigated the molecular mechanisms that drive the uPARAP-mediated cellular uptake of MBL and SP-D. We found that the uptake depends on residues within a protruding loop in the fibronectin type-II (FNII) domain of uPARAP that are also critical for collagen uptake. Importantly, however, we also identified FNII domain residues having an exclusive role in collectin uptake. We noted that these residues are absent in the related collagen receptor, the mannose receptor (MR or CD206), which consistently does not interact with collectins. We also show that the second C-type lectin-like domain (CTLD2) is critical for the uptake of SP-D, but not MBL, indicating an additional level of complexity in the interactions between collectins and uPARAP. Finally, we demonstrate that the same molecular mechanisms enable uPARAP to engage MBL immobilized on the surface of pathogens, thereby expanding the potential biological implications of this interaction. Our study reveals molecular details of the receptor-mediated cellular regulation of collectins and offers critical clues for future investigations into collectin biology and pathology.


Assuntos
Colectinas/metabolismo , Endocitose/fisiologia , Receptores Mitogênicos/genética , Animais , Células CHO , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Cricetulus , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lectinas Tipo C , Receptor de Manose , Lectina de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose , Glicoproteínas de Membrana/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular , Receptores de Colágeno/metabolismo , Receptores Mitogênicos/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo
17.
J Immunol ; 204(7): 1919-1928, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32094208

RESUMO

The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.


Assuntos
Lectinas/sangue , Animais , Células CHO , Linhagem Celular , Colectinas/metabolismo , Ativação do Complemento/fisiologia , Lectina de Ligação a Manose da Via do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Cricetulus , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos , Ficolinas
18.
J Cell Mol Med ; 24(6): 3572-3581, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32045104

RESUMO

Both Tamm-Horsfall protein (THP) and collectin-11 (CL-11) are important molecules in acute kidney injury (AKI). In this study, we measured the change of glycosylation of THP in patients with AKI after surgery, using MALDI-TOF MS and lectin array analysis. The amount of high-mannose and core fucosylation in patients with AKI were higher than those in healthy controls. In vitro study showed that THP could bind to CL-11 with affinity at 9.41 × 10-7  mol/L and inhibited activation of complement lectin pathway. The binding affinity decreased after removal of glycans on THP. Removal of fucose completely ablated the binding between the two proteins. While removal of high-mannose or part of the N-glycan decreased the binding ability to 30% or 60%. The results indicated that increase of fucose on THP played an important role via complement lectin pathway in AKI.


Assuntos
Injúria Renal Aguda/metabolismo , Colectinas/metabolismo , Uromodulina/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Galinhas , Eritrócitos/metabolismo , Feminino , Glicosilação , Hemólise , Humanos , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Polissacarídeos/metabolismo , Ligação Proteica , Ficolinas
19.
J Immunol ; 204(6): 1598-1606, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32041782

RESUMO

C1q/TNF-related protein (CTRP) 6 is a member of the CTRP protein family associated with the regulation of cellular and endocrine processes. CTRP6 contains collagen and globular structures, resembling the pattern recognition molecules (PRMs) of the classical and lectin complement pathways. We expressed human CTRP6 in Chinese hamster ovary cells and investigated the binding to different putative ligands (acetylated BSA [AcBSA], zymosan, mannan, and LPS from Escherichia coli and Salmonella as well as to the monosaccharides l-fucose, d-mannose, N-acetylglucosamine, N-acetylgalactosamine, and galactose). Furthermore, we investigated the binding of CTRP6 to various Gram-negative bacteria as well as PRMs and enzymes of the lectin complement pathway. We found that CTRP6 bound to AcBSA and to a lesser extent to zymosan. Using EDTA as chelating agent, we observed an increased binding to AcBSA, zymosan and the two strains of LPS. We detected no binding to mannan and BSA. We identified l-fucose as a ligand for CTRP6 and that it bound to certain enteroaggregative Escherichia coli and Pseudomonas aeruginosa isolates, whereas to other bacterial isolates, no binding was observed. CTRP6 did not appear to interact directly with the activating enzymes of the lectin pathway; however, we could show the specific recruitment of collectin-11 and subsequent initiation of the complement cascade through deposition of C4. In conclusion, our results demonstrate the binding of CTRP6 to a variety of microbial and endogenous ligands identifying CTRP6 as a novel human lectin and PRM of importance for complement recognition and innate immunity.


Assuntos
Antígenos de Bactérias/metabolismo , Colágeno/metabolismo , Colectinas/metabolismo , Complemento C4/metabolismo , Lectina de Ligação a Manose da Via do Complemento/imunologia , Animais , Antígenos de Bactérias/imunologia , Células CHO , Colágeno/genética , Colágeno/isolamento & purificação , Ativação do Complemento , Cricetulus , Escherichia coli/imunologia , Escherichia coli/metabolismo , Ligantes , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
20.
Dev Comp Immunol ; 102: 103486, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31473265

RESUMO

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity. CL-11, a protein similar to Mannose-binding lectin (MBL), plays significant role in the innate immune system in mammals and fish, serving as an initiator of the lectin pathway of complement activation. In this study, a CL-11 homolog (TfCol-11) was identified in roughskin sculpin (Trachidermus fasciatus), and its expression and role in immune responses were characterized. The open reading frame of TfCol-11 is 795 bp long, encoding a 264 amino acid polypeptide. The deduced amino acid sequence of this protein is highly homologous to sequences in other teleosts, and is similar to vertebrate CL-11, containing a canonical collagen-like region, a carbohydrate recognition domain, and a neck region. Recombinant TfCol-11 purified from Escherichia coli(E.coli) was able to bind to different microbes in a Ca2+-independent manner. Meanwhile, a 993 bp-long of partial MASP cDNA with a 96 bp 5' untranslated region (UTR) was also cloned from roughskin sculpin, containing 299 amino acids and consisting of three domains (CUB-EGF-CUB). qRT-PCR indicated that TfCol-11 and MASP mRNAs were predominately co-expressed in the liver. The temporal expression of TfCol-11 and MASP were both drastically up-regulated in the liver, skin, and blood by LPS challenge. Recombinant TfCol-11 purified from E.coli BL21(DE3) was able to agglutinate some bacteria in a Ca2+-dependent manner. In addition, an in vitro pull-down experiment demonstrated that TfCol-11 was able to bind to MASP, and in vivo experiments showed that TfCol-11 was associated with increased membrane attack complex (MAC) levels. It is therefore possible that TfCol-11 may plays a role in activating the complement system and in the defense against invading microorganisms in roughskin sculpin.


Assuntos
Colectinas/metabolismo , Ativação do Complemento , Proteínas de Peixes/metabolismo , Perciformes/imunologia , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Colectinas/química , Colectinas/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Fases de Leitura Aberta , Filogenia , Domínios Proteicos , Alinhamento de Sequência , Distribuição Tecidual
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