Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles.

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Ultrastructural Localization of Histidine-rich Glycoprotein in Skeletal Muscle Fibers: Colocalization With AMP Deaminase.

Histidine-rich glycoprotein (HRG) is a plasma protein synthesized by the liver. We have given the first evidence of a tissue localization of HRG demonstrating its presence in skeletal muscle, associated with the zinc enzyme AMP deaminase (AMPD1). Moreover, we have shown that muscle cells do not synthesize HRG, but they can internalize it from plasma. We have recently demonstrated by confocal laser scanning microscopy that in human skeletal muscle, HRG is mainly localized in the myofibrils, preferentially at the I-band of the sarcomere, in the sarcoplasm, and in the nuclei. Using transmission electron microscopy and immunogold analysis, we carried out this study on human and rat normal skeletal muscles with the purpose to deepen the ultrastructural localization of HRG in skeletal muscle fibers. The immunogold analysis evidenced the presence of HRG in the sarcomeres, mainly in the I-band and to a less extent in the A-band, in the heterochromatin of nuclei, and in the sarcoplasmic reticulum. The colocalization of HRG and skeletal muscle AMPD1 was also analyzed. A colabeling of HRG and AMPD1 was evident at sarcomeric, sarcoplasmic reticulum, and nuclear levels. The significance of these interesting and new results is discussed in this article.

Immunohistochemical localization of histidine-rich glycoprotein in human skeletal muscle: preferential distribution of the protein at the sarcomeric I-band.

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site.

We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (micro-aqua)(micro-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn(1) ion at the enzyme C-terminus, following the binding of AMP at the Zn(2) ion located at N-terminus of the enzyme. Thus, the two Zn ions in the AMPD metallocenter operate together as a single catalytic unit, but have independent function, one of them (Zn(1)) acting to polarize the nucleophile water molecule, whilst the other (Zn(2)) acts transiently as a receptor for an activating substrate molecule. The addition of fluoride to AMPD also abolishes the cooperative behaviour induced in the enzyme by the inhibitory effect of ATP at acidic pH that probably resides in the competition with the substrate for an adenine nucleotide specific regulatory site located in the Zn(2) ion binding region and which is responsible for the positive homotropic cooperativity behaviour of AMPD.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site.

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.

Immunohistochemical analysis of human skeletal muscle AMP deaminase deficiency. Evidence of a correlation between the muscle HPRG content and the level of the residual AMP deaminase activity.

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.

Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme.

The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.