RESUMO
Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Methods: Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. Results: The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1ß or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. Conclusions: SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.
Assuntos
Inflamação , Aparelho Lacrimal , Osteonectina , Animais , Camundongos , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Osteonectina/genética , Fatores EtáriosRESUMO
Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.
Assuntos
Lectinas Tipo C/química , Mucina-1/química , Sequência de Aminoácidos , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Calorimetria/métodos , Epitopos/metabolismo , Galactose , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Mucina-1/metabolismo , Ligação ProteicaRESUMO
Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Galectina 1/genética , Mucina-1/genética , Antígenos Glicosídicos Associados a Tumores/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Galectina 1/imunologia , Galectinas/genética , Galectinas/imunologia , Glicopeptídeos/genética , Glicopeptídeos/imunologia , Humanos , Mucina-1/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologiaRESUMO
CD147 is a widely expressed matrix metalloproteinase inducer involved in the regulation of cell migration. The high glycosylation and ability to undergo oligomerization have been linked to CD147 function, yet there is limited understanding on the molecular mechanisms behind these processes. The current study demonstrates that the expression of Golgi α1,2-mannosidase I is key to maintaining the cell surface organization of CD147 during cell migration. Using an in vitro model of stratified human corneal epithelial wound healing, we show that CD147 is clustered within lateral plasma membranes at the leading edge of adjacent migrating cells. This localization correlates with a surge in matrix metalloproteinase activity and an increase in the expression of α1,2-mannosidase subtype IC (MAN1C1). Global inhibition of α1,2-mannosidase I activity with deoxymannojirimycin markedly attenuates the glycosylation of CD147 and disrupts its surface distribution at the leading edge, concomitantly reducing the expression of matrix metalloproteinase-9. Likewise, treatment with deoxymannojirimycin or siRNA-mediated knockdown of MAN1C1 impairs the ability of the carbohydrate-binding protein galectin-3 to stimulate CD147 clustering in unwounded cells. We conclude that the mannose-trimming activity of α1,2-mannosidase I coordinates the clustering and compartmentalization of CD147 that follows an epithelial injury.
Assuntos
Basigina/metabolismo , Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Complexo de Golgi/enzimologia , Manosidases/metabolismo , Membrana Celular/metabolismo , Epitélio Corneano/citologia , Galectina 3/metabolismo , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
One of the main barriers to explaining the functional significance of glycan-based changes in cancer is the natural epitope heterogeneity found on the surface of cancer cells. To help address this knowledge gap, we focused on designing synthetic tools to explore the role of tumor-associated glycans of MUC1 in the formation of metastasis via association with lectins. In this study, we have synthesized for the first time a MUC1-derived positional scanning synthetic glycopeptide combinatorial library (PS-SGCL) that vary in number and location of cancer-associated Tn antigen using the "tea bag" approach. The determination of the isokinetic ratios necessary for the equimolar incorporation of (glyco)amino acids mixtures to resin-bound amino acid was determined, along with developing an efficient protocol for on resin deprotection of O-acetyl groups. Enzyme-linked lectin assay was used to screen PS-SGCL against two plant lectins, Glycine max soybean agglutinin and Vicia villosa. The results revealed a carbohydrate density-dependent affinity trend and site-specific glycosylation requirements for high affinity binding to these lectins. Hence, PS-SGCLs provide a platform to systematically elucidate MUC1-lectin binding specificities, which in the long term may provide a rational design for novel inhibitors of MUC1-lectin interactions involved in tumor spread and glycopeptide-based cancer vaccines.