Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears.
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ABSTRACT:
SUBMITTER: Schulz BL
PROVIDER: S-EPMC1222789 | biostudies-other | 2002 Sep
REPOSITORIES: biostudies-other
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Project description:Recent studies have shown that antibodies targeting Lewis x (Le(x)) antigen are a valuable tool in the isolation and identification of glycoproteins in plasma. A focus of this study was to determine whether sialylated Lewis x (sLe(x)) antigen carrying glycoproteins occur in human plasma and whether an antibody targeting this antigen could be used to isolate and identify glycoproteins bearing this antigen. An additional objective was to determine the degree to which proteins conjugated to Le(x) and sLe(x) antigens are similar in structure. A specific anti-sLe(x) antibody (anti-sLe(x)Ab), CHO-131, immobilized in an immunoaffinity column was used to select a set of specific sLe(x) bearing proteins from human plasma, after which they were identified by either of two analytical strategies. One approach was to further resolve the affinity selected proteins by reversed phase chromatography (RPC), tryptic digest the RPC fractions, and identify peptide fragments by MALDI-MS/MS. The second was to tryptic digest the affinity selected protein fraction, further resolve the tryptic fragments by RPC, and identify peptides from RPC fractions by MALDI-MS/MS. Histidine-rich glycoprotein, plasminogen, apolipoprotein A-I, vitronectin, proteoglycan-4, clusterin, Ig gamma-2 chain C region, Ig mu chain C region, and interalpha-trypsin inhibitor heavy chain H4 were found to change three folds or more in association with breast cancer. Fifty percent of the glycoproteins carrying either sLe(x) antigen from CHO-131 selection, Le(x) antigen from selection with TG-1 antibody, or both were found to be changed three folds or more in concentration in breast cancer plasma relative to controls.
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Project description:We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 µM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.
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Project description:In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.
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Project description:Over the last two decades, a large number of non-communicable/chronic disorders reached an epidemic level on a global scale such as diabetes mellitus type 2, cardio-vascular disease, several types of malignancies, neurological and eye pathologies-all exerted system's enormous socio-economic burden to primary, secondary, and tertiary healthcare. The paradigm change from reactive to predictive, preventive, and personalized medicine (3PM/PPPM) has been declared as an essential transformation of the overall healthcare approach to benefit the patient and society at large. To this end, specific biomarker panels are instrumental for a cost-effective predictive approach of individualized prevention and treatments tailored to the person. The source of biomarkers is crucial for specificity and reliability of diagnostic tests and treatment targets. Furthermore, any diagnostic approach preferentially should be noninvasive to increase availability of the biomaterial, and to decrease risks of potential complications as well as concomitant costs. These requirements are clearly fulfilled by tear fluid, which represents a precious source of biomarker panels. The well-justified principle of a "sick eye in a sick body" makes comprehensive tear fluid biomarker profiling highly relevant not only for diagnostics of eye pathologies but also for prediction, prognosis, and treatment monitoring of systemic diseases. One prominent example is the Sicca syndrome linked to a cascade of severe complications that include dry eye, neurologic, and oncologic diseases. In this review, protein profiles in tear fluid are highlighted and corresponding biomarkers are exemplified for several relevant pathologies, including dry eye disease, diabetic retinopathy, cancers, and neurological disorders. Corresponding analytical approaches such as sample pre-processing, differential proteomics, electrophoretic techniques, high-performance liquid chromatography (HPLC), enzyme-linked immuno-sorbent assay (ELISA), microarrays, and mass spectrometry (MS) methodology are detailed. Consequently, we proposed the overall strategies based on the tear fluid biomarkers application for 3P medicine practice. In the context of 3P medicine, tear fluid analytical pathways are considered to predict disease development, to target preventive measures, and to create treatment algorithms tailored to individual patient profiles.
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Project description:Human milk oligosaccharides (HMOS) are not digested in the proximal intestine. In distal intestine, HMOS collectively modify the microbiota, but the response of individual bacteria to individual components of the HMOS is not well defined. Here, each of 25 major isolates of the human intestinal microbiota was fed individual major fucosylated and sialylated HMOS in anaerobic culture. This allowed for an assessment of the influence of specific HMOS on the growth and metabolic products of individual microbiota bacteria. Most Bifidobacteria spp. and Bacteroides spp. grew, induced ?-L-fucosidase activity, and produced abundant lactate or short-chain fatty acids (SCFAs) when fed 2'-fucosyllactose (2'-FL), 3-FL, and lactodifucotetraose (LDFT). Lactobacillus delbrueckii ATCC7830, Enterococcus faecalis ATCC19433, and Streptococcus thermophilus ATCC19258 exhibited slight growth, pH reduction, and lactate production when supplemented with 2'-FL or 3-FL, but not LDFT. Supplementation with 3'-sialyllactose (3'-SL) and 6'-SL promoted moderate growth of Bifidobacterium longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, Bacteroides vulgatus ATCC8482, and B. thetaiotaomicron ATCC29148; accordingly, these bacteria exhibited greater neuraminidase activity and produced copious lactate, SCFA, or both. Lactobacillus delbrueckii ATCC7830 also consumed 6'-SL. In contrast, Clostridium spp., L. rhamnosus ATCC53103, E. faecalis ATCC29200, Staphylococcus spp., Enterobacter spp., and Escherichia coli K12 did not consume milk oligosaccharides nor produce appreciable acidic fermentation products. Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated milk oligosaccharides most strongly stimulating key species of mutualist symbionts. This suggests strategies for treating dysbiosis of the microbiota and associated inflammatory disorders.
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Project description:PurposeTo determine the source(s) of vitamin D in tear fluid and examine the expression of the endocytic proteins and putative vitamin D transporters megalin and cubilin in lacrimal and Harderian glands.MethodsWild-type, heterozygous, and vitamin D receptor (VDR) knockout C57BL/6 mice were used, with a subset of knockout mice fed a replenishment diet for some studies. Mouse lacrimal and Harderian glands from each group were used to measure megalin and cubilin by RT-PCR, Western blot, and immunohistochemistry. New Zealand white rabbits were used to collect lacrimal and accessory gland fluid for vitamin D mass spectroscopy measurements.ResultsTen-week-old knockout mice were significantly (P < 0.05) smaller than wild-type mice. Real-time PCR and Western blot showed decreased expression of megalin and cubilin in select VDR knockout mouse groups. Immunohistochemistry showed apical duct cell megalin staining and weaker megalin staining in VDR knockout mice compared with controls. Vitamin D2 was more prevalent in rabbit lacrimal and accessory gland fluid than vitamin D3, and greater amounts of Vitamin D2 were found in in tear fluid obtained directly from lacrimal and accessory glands as compared with plasma concentrations.ConclusionsThis is the first study to demonstrate the presence of megalin and cubilin in lacrimal and accessory glands responsible for producing tear fluid. The results strengthen the hypothesis that megalin and cubilin are likely involved in the secretory pathway of vitamin D into tear fluid by the duct cells.
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Project description:Anterior cruciate ligament (ACL) tears occur in isolation or in combination with other intra-articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of the injured ACL is unknown. Here, we tested the hypothesis that the biological response of the ACL to injury varies based on the presence or absence of concomitant meniscus tear. RNA-seq analysis was performed on 28 ACL tears remnants (12 isolated, 16 combined). 16,654 transcripts were differentially-expressed between isolated and combined injury groups at false-discovery-rate of 0.05. Due to the large number of differentially expressed transcripts, we undertook an Ensembl approach to discover features that acted as hub-genes that did not necessarily have large fold-changes or high statistical significance, but instead had high biological significance. Our data revealed a negatively-correlated turquoise-module containing 5960 transcripts (down-regulated in combined injury) and a positively-correlated blue-module containing 2260 transcripts (up-regulated in combined injury). TNS1, MEF2D, NOTCH3, SOGA1, and MLXIP were highly-connected hub-genes in the turquoise-module and SCN2A, CSMD3, LRC44, USH2A, and LRP1B were critical hub-genes in the blue-module. Transcripts in the turquoise-module were associated with biological-adhesion, actin-filament organization, cell-junction assembly, and cell-matrix adhesion. The blue-module transcripts were enriched for neuron-migration and exocytosis-regulation. These findings indicate a loss of healing and gain of neurogenic signaling in combined ACL and meniscus tears, suggesting they have diminished potential for repair. The biological response of the ACL to injury could have implications for the healing potential of the ligament and the long-term health of the knee.
2018-01-23 | GSE109419 | GEO
Project description:To understand the pathophysiology of dry eye disease (DED), it is necessary to characterize proteins in the ocular surface fluids, including tear fluid (TF) and lacrimal fluid (LF). There have been several reports of TF proteomes, but few proteomic studies have examined LF secreted from the lacrimal gland (LG). Therefore, we characterized the proteins constituting TF and LF by liquid chromatography mass spectrometry. TF and LF were collected from patients with non-Sjögren syndrome DED and from healthy subjects. Through protein profiling and label-free quantification, 1165 proteins from TF and 1448 from LF were identified. In total, 849 proteins were present in both TF and LF. Next, candidate biomarkers were verified using the multiple reaction monitoring assay in both TF and LF of 17 DED patients and 17 healthy controls. As a result, 16 marker proteins were identified (fold-change?>?1.5, p-value?<?0.05), of which 3 were upregulated in TF and 8 up- and 5 down-regulated in LF. In conclusion, this study revealed novel DED markers originating from the LG and tears by in-depth proteomic analysis and comparison of TF and LF proteins.
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Project description:BACKGROUND:Depression affects approximately 7.1% of the United States population every year and has an annual economic burden of over $210 billion dollars. Several recent studies have sought to investigate the pathophysiology of depression utilizing focused cerebrospinal fluid (CSF) and serum analysis. Inflammation and metabolic dysfunction have emerged as potential etiological factors from these studies. A dysregulation in the levels of inflammatory proteins such as IL-12, TNF, IL-6 and IFN-? have been found to be significantly correlated with depression. METHODS:CSF samples were obtained from 15 patients, seven with major depressive disorder and eight age- and gender-matched non-psychiatric controls. CSF protein profiles were obtained using quantitative mass spectrometry. The data were analyzed by Progenesis QI proteomics software to identify significantly dysregulated proteins. The results were subjected to bioinformatics analysis using the Ingenuity Pathway Analysis suite to obtain unbiased mechanistic insight into biologically relevant interactions and pathways. RESULTS:Several dysregulated proteins were identified. Bioinformatics analysis indicated that the potential disorder/disease pathways include inflammatory response, metabolic disease and organismal injury. Molecular and cellular functions that were affected include cellular compromise, cell-to-cell signaling & interaction, cellular movement, protein synthesis, and cellular development. The major canonical pathway that was upregulated was acute phase response signaling. Endogenous upstream regulators that may influence dysregulation of proinflammatory molecules associated with depression are interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), oncostatin M, PR domain zinc finger protein 1 (PRDM1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). CONCLUSIONS:The proteome profiling data in this report identifies several potential biological functions that may be involved in the pathophysiology of major depressive disorder. Future research into how the differential expression of these proteins is involved in the etiology and severity of depression will be important.
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Project description:Sialic-acid-mediated interactions play critical roles on the cell surface, providing an impetus for the development of methods to study this important monosaccharide. In particular, photo-cross-linking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic-acid-recognizing proteins via cross-linking. However, natural sialic acids also present on the cell surface compete with photo-cross-linking sialic acids in binding events, limiting cross-linking yields. In order to improve the utility of one such photo-cross-linking sialic acid, SiaDAz, we examined a number of sialidases, enzymes that remove sialic acids from glycoconjugates, to find one that would cleave natural sialic acids but remain inactive toward SiaDAz. Using this sialidase, we improved SiaDAz-mediated cross-linking of an antisialyl Lewis X antibody and of endoglin. This protocol can be applied generally to sialic-acid-mediated interactions and will facilitate identification of sialic acid binding partners.
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