Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation.

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae).

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30-50°C, and 0.05 M Ca(2+) or Mg(2+); and has a Km of 0.5 µg/ml and a Vmax of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a Km of 0.93 mg/ml and a Vmax of 1.63.

Chicken-type lysozyme in channel catfish: expression analysis, lysozyme activity, and efficacy as immunostimulant against Aeromonas hydrophila infection.

To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.

Proteomics and multivariate modelling reveal sex-specific alterations in distinct regions of human carotid atheroma.

BACKGROUND: Atherosclerotic lesions are comprised of distinct regions with different proteomic profiles. Men and women develop differences in lesion phenotype, with lesions from women generally being more stable and less prone to rupture. We aimed to investigate the differences in proteomic profiles between sexes, including distinct lesion regions, to identify altered proteins that contribute to these differences observed clinically. METHODS: Carotid endarterectomy samples (ten men/ten women) were obtained, and intraplaque biopsies from three distinct regions (internal control, fatty streak and plaque) were analysed by tandem-mass spectrometry. Multivariate statistical modelling, using orthogonal partial least square-discriminant analysis, was used to discriminate the proteomes between men and women. RESULTS: Multivariate discriminant modelling revealed proteins from 16 functional groups that displayed sex-specific associations. Additional statistics revealed ten proteins that display region-specific alterations when comparing sexes, including proteins related to inflammatory response, response to reactive oxygen species, complement activation, transport and blood coagulation. Transport protein afamin and blood coagulation proteins antithrombin-III and coagulation factor XII were significantly increased in plaque region from women. Inflammatory response proteins lysozyme C and phospholipase A2 membrane-associated were significantly increased in plaque region from men. Limitations with this study are the small sample size, limited patient information and lack of complementary histology to control for cell type differences between sexes. CONCLUSIONS: This pilot study, for the first time, utilises a multivariate proteomic approach to investigate sexual dimorphism in human atherosclerotic tissue, and provides an essential proteomic platform for further investigations to help understand sexual dimorphism and plaque vulnerability in atherosclerosis.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation.

PURPOSE: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. MATERIALS AND METHODS: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. RESULTS: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CONCLUSION: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Genomic organization and evolution of ruminant lysozyme c genes.

Ruminant stomach lysozyme is a long established model of adaptive gene evolution. Evolution of stomach lysozyme function required changes in the site of expression of the lysozyme c gene and changes in the enzymatic properties of the enzyme. In ruminant mammals, these changes were associated with a change in the size of the lysozyme c gene family. The recent release of near complete genome sequences from several ruminant species allows a more complete examination of the evolution and diversification of the lysozyme c gene family. Here we characterize the size of the lysozyme c gene family in extant ruminants and demonstrate that their pecoran ruminant ancestor had a family of at least 10 lysozyme c genes, which included at least two pseudogenes. Evolutionary analysis of the ruminant lysozyme c gene sequences demonstrate that each of the four exons of the lysozyme c gene has a unique evolutionary history, indicating that they participated independently in concerted evolution. These analyses also show that episodic changes in the evolutionary constraints on the protein sequences occurred, with lysozyme c genes expressed in the abomasum of the stomach of extant ruminant species showing the greatest levels of selective constraints.

Isolation and characterization of a novel angiotensin-converting enzyme-inhibitory tripeptide from enzymatic hydrolysis of soft-shelled turtle (Pelodiscus sinensis) egg white: in vitro, in vivo, and in silico study.

In this study, a novel angiotensin-converting enzyme (ACE)-inhibitory tripeptide (IVR) was isolated and identified from unfertilized soft-shelled turtle egg white (SSTEW). The IC50 value of IVR was measured in vitro as low as 0.81 ± 0.03 µM, and its inhibition type was suggested as competitive according to the Lineweaver-Burk plot. This peptide can be generated from either thermolysin followed by trypsin digestion (two stages) or only trypsin digestion (one stage). Quantitative LC-MS/MS analysis indicated that two-stage digestion gave 3.14 ± 0.17 mg of IVR from 1 g of SSTEW, better than that from one-stage digestion (1.31 ± 0.12 mg). In vivo antihypertensive activity of the tripeptide IVR after single oral administration (0.1 and 1 mg/kg of body weight) led to a significant reduction in systolic blood pressure 2-4 h after administration in spontaneously hypertensive rats. In addition, the binding mechanism of IVR has been rationalized through docking simulations using the testicular ACE (tACE)-lisinopril complex at 2 Å resolution (PDB 108A ). The best docking pose was located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, an effective hypertensive synthetic drug. The degree of inhibition of this peptide correlated with the H-bond interaction between the C-terminal of IVR and Lys511 and Tyr520 residues of tACE, a significant inhibitor registration for lisinopril. This study illustrated that IVR behaves as a transition-state analogue inhibitor and is useful in therapeutic intervention for blood pressure control. To the best of our knowledge, this is the first report of an efficient ACE-inhibitory tripeptide generated from the unfertilized egg of soft-shelled turtle.

Vitelline membrane outer layer 1 homolog interacts with lysozyme C and promotes the stabilization of tear film.

PURPOSE: The aim of this study was to explore the possible interactions between vitelline membrane outer layer 1 homolog (VMO1) and other tear proteins and to determine the function of VMO1 in tear fluid. METHODS: Interactions between recombinant human VMO1 and several abundant tear proteins were determined by dot blot, His pull-down, immunoprecipitation, and Western blot assays, as well as by computer-assisted prediction and modeling of molecular interactions. Kirby-Bauer antibiotic testing was performed to determine whether VMO1 possesses antimicrobial activity. Tear samples were collected from dry eye patients and from healthy controls. The role of VMO1 in maintaining the stability of tear film was investigated by measurement of contact angles on Teflon, tear break-up time (TBUT) and the time-dependent reduction in tear film integrity in mice. RESULTS: Vitelline membrane outer layer 1 homolog showed an interaction with lysozyme C (LYSC) in the dot-blot, His pull-down, and immunoprecipitation assays. Vitelline membrane outer layer 1 homolog revealed no zones of growth inhibition of standard strains of Staphylococcus aureus and Escherichia coli. Tears presented smaller contact angles on Teflon surfaces after the addition of VMO1 (P<0.05). Vitelline membrane outer layer 1 homolog-treated mice showed longer TBUTs (P<0.05). Tear films from VMO1-treated mice maintained their integrity for longer periods of time than tear films from the control group, and this effect was dose-dependent. CONCLUSIONS: Vitelline membrane outer layer 1 homolog interacts with LYSC and has positive effects on the stabilization of tear film.

Proteome of human plasma very low-density lipoprotein and low-density lipoprotein exhibits a link with coagulation and lipid metabolism.

Apart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL- and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti- microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 - dyslipidaemia, 2 - atherosclerosis and vascular disease, and 3 - coagulation disorders.

A novel angiotensin converting enzyme inhibitory peptide derived from proteolytic digest of Chinese soft-shelled turtle egg white proteins.

In this study, soft-shelled turtle (Pelodiscus sinensis) egg white (SSTEW) proteins were digested by thermolysin and the resulting small peptides were further fractionated by reverse phase chromatography. Peptides with angiotensin I-converting enzyme inhibitory (ACEI) activity from these fractions were screened. A lysozyme-derived peptide, IW-11, from the fraction with the most effective ACEI was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its purified form showed effective ACEI activity in vitro (IC50=4.39±0.31µM). The Lineweaver-Burk plots indicated that the inhibition towards ACE caused by this peptide is a competitive inhibition. The molecular docking study further revealed that the ACEI activity of IW-11 is mainly attributed to the formation of hydrogen bonds between the N-terminal residue of IW-11 and the S1 pocket (Ala354 and Tyr523) and the S2' region (His513 and His353) of ACE. Moreover, the digestion parameters were further optimized and the target peptide (82% purity) was readily obtained (15% yield) without any cumbersome purification procedure. Notably, lysozyme C is the most abundant protein in SSTEW, which implies that an efficient production of this ACEI peptide from SSTEW is promising. BIOLOGICAL SIGNIFICANCE: Inhibition of ACE has proven to be an effective strategy in prevention and treatment of hypertension and related diseases. Unlike typical synthetic ACE inhibitors which exert well described side effects, food-derived peptides with ACE inhibitory activity may be safer alternatives for hypertension treatment. In this study, we comprehensively identified peptides derived from SSTEW digest using a proteomic approach. IW-11, which is derived from lysozyme, the most abundant protein in SSTEW, showed remarkable inhibition towards ACE. This peptide has been demonstrated to have a competitive inhibitory property which is able to bind to ACE active site and found to be a true inhibitor against ACE according to Lineweaver-Burk plots. Using an optimized thermolysin condition, IW-11 can be readily obtained without any complex purification step, which will benefit its further application to prevention or treatment of hypertension.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

PURPOSE: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. MATERIALS AND METHODS: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. RESULTS: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CONCLUSION: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

PURPOSE: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. MATERIALS AND METHODS: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. RESULTS: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CONCLUSION: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.