Extracellular vesicles (EVs) harbor thousands of proteins that hold promise for biomarker development. Usually difficult to purify, EVs in urine are relatively easily obtained and have demonstrated ...efficacy for kidney disease prediction. Herein, we further characterize the proteome of urinary EVs to explore the potential for biomarkers unrelated to kidney dysfunction, focusing on Parkinson's disease (PD).
Using a quantitative mass spectrometry approach, we measured urinary EV proteins from a discovery cohort of 50 subjects. EVs in urine were classified into subgroups and EV proteins were ranked by abundance and variability over time. Enriched pathways and ontologies in stable EV proteins were identified and proteins that predict PD were further measured in a cohort of 108 subjects.
Hundreds of commonly expressed urinary EV proteins with stable expression over time were distinguished from proteins with high variability. Bioinformatic analyses reveal a striking enrichment of endolysosomal proteins linked to Parkinson's, Alzheimer's, and Huntington's disease. Tissue and biofluid enrichment analyses show broad representation of EVs from across the body without bias towards kidney or urine proteins. Among the proteins linked to neurological diseases, SNAP23 and calbindin were the most elevated in PD cases with 86% prediction success for disease diagnosis in the discovery cohort and 76% prediction success in the replication cohort.
Urinary EVs are an underutilized but highly accessible resource for biomarker discovery with particular promise for neurological diseases like PD.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. ...Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood.
Objective
To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities.
Methods
Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain.
Results and Conclusions
We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide‐induced plasma coagulation. Wild‐type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide‐induced auto‐activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+‐binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.
Full text
Available for:
FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen ...capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.
Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.
At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell ...(L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.
The excessive intake of sodium (Na) and insufficient intake of potassium (K) are major concerns in the prevention of hypertension. Using low-Na/K seasonings (reducing 25% of the NaCl and adding K ...salt) may improve the dietary Na/K ratio and help prevent hypertension. To devise an intervention study using low-Na/K seasonings at a company cafeteria, we calculated the Na and K contents of the meals served at the cafeteria and estimated changes in the intakes when suitable low-Na/K seasonings were used. We also considered using milk as a good source of K. We used an ingredient list of a company cafeteria and calculated Na and K contents in each dish. The average amounts of NaCl and K per use were 5.04 g and 718 mg, respectively. Seasonings contributed 70.9% of the NaCl. With the use of low-Na/K seasonings, an estimated reduction in NaCl of 0.8 g/day and an estimated increase in K of 308 mg/day was achieved. With an additional serving (200 mL) of milk, NaCl was reduced by 0.57 g/day and K was increased by 610 mg/day, with an overall decrease in the dietary Na/K ratio from 3.20 to 2.40. The use of low-Na/K seasonings and dairy may improve the dietary Na/K ratio among cafeteria users and help prevent hypertension.
ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol ...mannosides (PIMs) by glycan microarray and NMR. Pathogen‐related glycan microarray analysis identified phosphatidylinositol mono‐ and di‐mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical‐shift perturbation experiments with uniformly 15N‐labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p.
Mycobacterial glycolipids trapped by a human lectin: Human lectin ZG16p is expressed in the gastrointestinal mucosa, but its function is unclear. Glycan microarray revealed that human ZG16p interacts with mycobacterial phosphatidylinositol mannosides (PIMs). Precise NMR interaction analysis established the PIM‐glycan–ZG16p binding mode.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue‐specific KrasG12D‐knockin mouse model of pancreatic cancer that consistently forms precancerous ...lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top‐associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN‐γ, VEGF and GM‐CSF were significantly increased in serum from the KrasG12D animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early‐stage neoplasia.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The common FcRγ, an immunoreceptor tyrosine‐based activation motif (ITAM)‐ containing adaptor protein, associates with multiple leukocyte receptor complexes and mediates signal transduction through ...the ITAM in the cytoplasmic domain. The presence of multiple serine and threonine residues within this motif suggests the potential for serine/threonine phosphorylation in modulating signaling events. Single‐site mutational analysis of these residues in RBL‐2H3 cells indicates that each may contribute to net FcRγ‐mediated signaling, and mass spectrometry of WT human FcRγ from receptor‐stimulated cells shows consistent preferential phosphorylation of the serine residue at position 51. Immunoblot analysis, mass spectrometry, and mutational analyses showed that phosphorylation of serine 51 in the 7‐residue spacer between the 2 YxxL sequences regulates FcRγ signaling by inhibiting tyrosine phosphorylation at the membrane proximal Y47 position of the ITAM, but not phosphorylation at position Y58. This inhibition results in reduced Syk recruitment and activation. With in vitro kinase assays, PKC‐δ and PKA show preferential phosphorylation of S51. Serine/threonine phosphorylation of the FcRγ ITAM, which functions as an integrator of multiple signaling elements, may explain in part the contribution of variants in PKC‐δ and other PKC isoforms to some autoimmune phenotypes.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Various enzyme-conjugated probes have been widely used for detection of specific interactions between biomolecules. In the case of glycan-protein interaction, horseradish peroxidase (HRP)-conjugated ...glycoproteins (HRP-GPs) are useful for the detection of carbohydrate-binding activity of plant and animal lectins. In this chapter, a typical solid-phase assay of the carbohydrate-binding activity of Sophora japonica agglutinin I, a Gal/GalNAc-specific lectin, using HRP-conjugated asialofetuin is described. HRP-GPs are versatile tools for probing lectin activities in crude extracts, screening many samples at one time, and applicable not only for solid-phase binding assays but also samples which are dot- or Western-blotted onto the membrane.
HMMC‐1 is a human monoclonal antibody that reacts with a fucosylated and extended core 1 O‐glycan, Fucα1‐2Galβ1‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐Ser/Thr, as an epitope. In the present study, we examined the ...effects of HMMC‐1 on cell proliferation of two human uterine endometrial cancer cell lines, HEC8 and HEC9, to investigate the role of glycoproteins bearing the HMMC‐1 epitope in cancer progression. HEC9 cells expressed high levels of the HMMC‐1 epitope, but HMMC‐1 reactivity was hardly detected in HEC8 cells. In a mouse model of lymph node metastasis using orthotopic implantation, HEC8 and HEC9 showed low (10%) and high (80%) metastatic potency, respectively. Growth of HEC9, but not HEC8, was remarkably inhibited by addition of HMMC‐1 to the culture medium. Cell cycle analysis and expression analysis showed that HMMC‐1 treatment increased the G1 phase population of HEC9 cells and induced cyclin‐dependent kinase inhibitors p16 and p21. Two glycoproteins, 97 and 137 kDa, with a strong reactivity to HMMC‐1 were purified, and the 97‐kDa glycoprotein was identified as CD166, an immunoglobulin superfamily cell adhesion molecule assumed to be involved in cancer metastasis. CD166 gene‐silencing dramatically reduced HMMC‐1 epitope expression and growth in HEC9 cells, indicating that CD166 is the primary glycoprotein presenting the HMMC‐1 epitope in HEC9 cells. Collectively, HMMC‐1 might arrest the cell cycle in the G1 phase by binding to O‐glycans on the CD166 expressed in HEC9 cells, raising the possibility that HMMC‐1 extensively inhibits invasive growth of HMMC‐1 epitope‐positive uterine endometrial cancer cells by targeting the cancer‐associated form of CD166. (Cancer Sci 2013; 104: 62–69)
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
PDF
