Linker histone‐like proteins in Muscovy duck (Cairina moschata L) erythrocyte chromatin

Abstract

Linker Histone-Like proteins (LHL1 and LHL2) were identified within a linker histone complement of Muscovy duck erythrocyte chromatin. Polyacrylamide gel electrophoretic patterns of N-bromosuccinimide-cleaved LHL products as well as liquid chromatography-electrospray-ion trap mass spectrometry analyses of trypsin-digested LHL peptides revealed structural similarity of LHL1 to histone H5 and between LHL2 and histone H1 subtypes. Since the LHL proteins were stable in the presence of 2-mercaptoethanol and dithiothreitol that reduce disulfide bonds, it appeared unlikely that this doublet was a thiol-derived product of linker histones. A loss of LHL1, with a concomitant maintenance of LHL2 after treatment with dilute alkali, seems to suggest that they might represent disparate protein conjugates resulting from linker histone modifications through ester linkages.

Polymorphism of histone H1.c’ in the population of Muscovy duck (Cairina moschata L.): a link between histone H1.c’ allelic variants and ADP-ribosylation of histone H1 subtypes

“…Subsequently, this protein was identified as a complex, named LHL, consisting of histone H1 subtypes (H1.b and H5) linked to the ADPribose. In two-dimensional polyacrylamide gel, it is visible as a slowly migrating protein spot that forms a dimer in one-dimensional SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel (Kowalski et al 2009). Such protein doublets with retarded electrophoretic mobility were identified earlier as poly-ADPribosylated histone H1 forms (Poirier et al 1982;Niedergang et al 1985), so the presence of LHL conjugate in the Muscovy duck H1 histones may correspond to the effect of poly(ADP-ribosylation).…”

High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis: A Tool for Identification of Polymorphic and Modified Linker Histone Components

“…Usually, the 2D-PAGE patterns of linker histones represented a diagonal arrangement of non-overlapping protein spots. The use of this technique was also helpful for detection of allelic and/or posttranslationally modified forms of linker histone subtypes undetectable in the first dimension acetic acid-urea polyacrylamide gel and visible as single or double protein spots when resolved in the second dimension in the polyacrylamide gel containing SDS (Kowalski et al, 2009). …”

“…They can be visible as small protein spots accompanying abundant non-modified parental proteins, just like LHL (linker histone-like) adducts of linker histone subtype probably with ADP-ribose ( Fig. 6 and 9) (Kowalski et al, 2009) or as protein band(s) moving faster than main protein component, like phosphorylated forms of murine histone H1 (Lennox et al, 1982). It seems likely that additional protein component a1 m accompanying allelic variant H1.a1 (Fig.…”

“…Two-dimensional PAGE can also be used as a preparatory step for protein identification in gel pieces by mass spectrometry (Fig. 9) (Górnicka-Michalska et al, 2006;Kowalski et al, 2009) or for gathering a greater amount of concentrated protein to protease cleavage (Górnicka-Michalska et al, 2006;Kowalski et al, 2011) (Fig. 10) and microsequencing (Górnicka-Michalska et al, 2006).…”

“…This electrophoretic system is relatively straightforward and reproducible. Beside qualitative and quantitative applications (Kowalski et al, 2011a(Kowalski et al, , 2011b for protein detection and subsequent abundance estimation, the 2D-PAGE-like method may also be used on a preparative scale to enable gathering and purification of appropriate amount of the resolved protein for subsequent structural analyses including chemical cleavage and enzymatic digestion Pałyga & Neelin, 1998;Pałyga et al, 2000;Kowalski et al, 2011aKowalski et al, , 2011b as well as amino acid sequencing (Górnicka-Michalska et al, 2006) and mass spectrometry analysis (Kowalski et al, 2009).…”