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Biomedical subjects

Juan Perez-Vilar

Publications and source records attributed to Juan Perez-Vilar.

8 recordsLinked to original sources
PubMed2006

Mucin granule intraluminal organization.

Mucus secretions have played a central role in the evolution of multicellular organisms, enabling adaptation to widely differing environments. In vertebrates, mucus covers and protects the epithelial cells in the respiratory, gastrointestinal, urogenital, visual, and auditory systems, amphibian's epidermis, and the gills in fishes. Deregulation of mucus production and/or composition has important consequences for human health. For example, mucus obstruction of small airways is observed in chronic airway diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis. The major protein component in the mucus is a family of large, disulfide-bonded glycoproteins known as gel-forming mucins. These proteins are accumulated in large, regulated secretory granules (the mucin granules) that occupy most of the apical cytoplasm of specialized cells known as mucous/goblet cells. Since mucin oligomers have contour dimensions larger than the mucin granule average diameter, the question arises how these highly hydrophilic macromolecules are organized within these organelles. I review here the intraluminal organization of the mucin granule in view of our knowledge on the structure, biosynthesis, and biophysical properties of gel-forming mucins, and novel imaging studies in living mucous/goblet cells. The emerging concept is that the mucin granule lumen comprises a partially condensed matrix meshwork embedded in a fluid phase where proteins slowly diffuse.

Animals↗
PubMed2005

Mucin granule intraluminal organization in living mucous/goblet cells. Roles of protein post-translational modifications and secretion.

Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t(1/2)), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t((1/2)) while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule.

Animals↗
PubMed2005

Mucin granules are in close contact with tubular elements of the endoplasmic reticulum.

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

Cell Line, Tumor↗
PubMed2005

Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia.

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.

Adolescent↗
PubMed2005

pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells.

To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, accumulated in the granules together with native MUC5AC. Inhibition of protein synthesis or disorganization of the Golgi complex did not result in diminished intragranular SHGFP-MUC5AC/CK signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated that SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. Analysis of fluorescence recovery after photobleaching suggested that the intragranular SHGFP-MUC5AC/CK mobile fraction and mobility were significantly lower than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (approximately 20%) the intragranular SHGFP-MUC5AC/CK mobile fraction. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intraluminal distribution of the fluorescent fusion protein. These results are consistent with a model of mucin granule intraluminal organization with two phases: a mobile phase in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate and an immobile phase or matrix in which proteins are immobilized by noncovalent pH-dependent interactions. An intraluminal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.

Adenosine Triphosphatases↗
PubMed2004

Reevaluating gel-forming mucins' roles in cystic fibrosis lung disease.

The existence of mucus plugs, containing mucins, bacteria, and neutrophils, blocking the lower airways in the lung of cystic fibrosis (CF) patients has raised the possibility that production of "abnormal" mucins is a critical characteristic of this disease. The molecular nature, if any, of this abnormality is unknown. Recent studies suggest that CF lung disease progression is characterized by an early phase in which airway surface liquid (ASL) increased dehydration is accompanied by altered pH and levels of reduced glutathione (GSH). In a later phase, bacterial infection and neutrophil invasion lead to increased ASL of concentrations myeloperoxidase and hypochlorous acid (HOCl). Independent studies indicate that gel-forming mucins, the key components of airway mucus, form disulfide-linked polymers through a pH-dependent, likely self-catalyzed mechanism. In this article, we present the hypothesis that increased mucus concentration (dehydration) and altered pH, and levels of GSH, myeloperoxidase, and/or HOCl result in the extracellular formation of additional interchain bonds among airway mucins. These novel interactions would create an atypical mucin network with abnormal viscoelastic and adhesive properties.

Bacterial Infections↗
PubMed2004

C-Mannosylation of MUC5AC and MUC5B Cys subdomains.

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.

Amino Acid Sequence↗