scholarly journals Histological and Top-Down Proteomic Analyses of the Visual Pathway Using the Cuprizone Demyelination Model

2021 ◽  
Author(s):  
Mohammed S. M. Almuslehi ◽  
Monokesh K. Sen ◽  
Peter J. Shortland ◽  
David A. Mahns ◽  
Jens R. Coorssen
Keyword(s):  
Animal Models ◽  
Visual Pathway ◽  
Bioinformatic Analysis ◽  
Mass Spectrometry Analysis ◽  
Literature Mining ◽  
Top Down ◽  
Spectrometry Analysis ◽  
Cytoskeleton Organization ◽  
Adult Mice ◽  
Liquid Chromatography Tandem Mass

Abstract A change in visual perception is a frequent early symptom of multiple sclerosis (MS), the pathoetiology of which remains unclear. Following a slow demyelination process caused by 12 weeks of low-dose (0.1%) cuprizone (CPZ) consumption, histology and proteomics were used to investigate components of the visual pathway in young adult mice. Histological investigation did not identify demyelination or gliosis in the optic tracts, pretectal nuclei, superior colliculi, lateral geniculate nuclei or visual cortices. However, top-down proteomic assessment of the optic nerve/tract revealed a significant change in the abundance of 34 spots in high-resolution 2D gels. Subsequent liquid chromatography-tandem mass spectrometry analysis identified alterations in 75 proteoforms. Literature mining revealed the relevance of these proteoforms in terms of proteins previously implicated in animal models and human MS. Importantly, 24 proteoforms were not previously described in any animal models of MS or MS itself. Bioinformatic analysis indicated involvement of these proteoforms in cytoskeleton organization, metabolic dysregulation, protein aggregation, and axonal support. Collectively, these results indicate that continuous CPZ-feeding, which evokes a slow demyelination, results in proteomic changes that precede any clear histological changes in the visual pathway and that these proteoforms may be potential early markers of degenerative demyelinating conditions.

scholarly journals NUP62: the target of an anti-sperm auto-monoclonal antibody during testicular development

Reproduction ◽  
2019 ◽  
Vol 158 (6) ◽  
pp. 503-516
Author(s):  
Risako Oda-Sakurai ◽  
Hiroshi Yoshitake ◽  
Yoshiki Miura ◽  
Saiko Kazuno ◽  
Takashi Ueno ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Germ Cells ◽  
Early Embryo ◽  
Mass Spectrometry Analysis ◽  
Testicular Development ◽  
Reproductive Process ◽  
Spectrometry Analysis ◽  
Adult Mice ◽  
Liquid Chromatography Tandem Mass ◽  
Epididymal Spermatozoa

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.

Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes

2016 ◽  
Vol 107 (1) ◽  
pp. 66-76 ◽  
Author(s):  
M.I. Boguś ◽  
W. Wieloch ◽  
M. Ligęza-Żuber
Keyword(s):  
Entomopathogenic Fungus ◽  
Galleria Mellonella ◽  
Elongation Factor ◽  
Peptide Sequence ◽  
Mass Spectrometry Analysis ◽  
Sequence Coverage ◽  
Spectrometry Analysis ◽  
Conidiobolus Coronatus ◽  
Liquid Chromatography Tandem Mass

AbstractCoronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC–MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva−1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

Enhancing proteasomal processing improves survival for a peptide vaccine used to treat glioblastoma

2021 ◽  
Vol 13 (598) ◽  
pp. eaax4100
Author(s):  
Mario Fidanza ◽  
Puja Gupta ◽  
Anin Sayana ◽  
Varun Shanker ◽  
Svenja-Maria Pahlke ◽  
...  
Keyword(s):  
Immune Response ◽  
Mhc Class I ◽  
Peptide Vaccine ◽  
Tumor Model ◽  
Class I ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Tandem Mass Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Anticancer Vaccine

Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in several trials for glioblastoma. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model in mice. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti–PD-1 therapy cured 45% of the Y6-pepVIII–vaccinated mice but was ineffective for pepVIII-treated mice. Liquid chromatography–tandem mass spectrometry analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant in Y6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments may be a component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. We hypothesize that the immune response to a vaccine represents the collective contribution from multiple PCPS and linear products. Our work suggests a strategy to increase proteasomal processing of a vaccine that results in an augmented immune response and enhanced survival in mice.

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