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.
Perez-Vilar
Juan
J
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA. juan_vilar@med.unc.edu
Mabolo
Raean
R
McVaugh
Cheryl T
CT
Bertozzi
Carolyn R
CR
Boucher
Richard C
RC
D000818:Animals; D017209:Apoptosis; D002241:Carbohydrates / Q000737:chemistry; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002704:Chlorates / Q000494:pharmacology; D004058:Diffusion; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D006031:Glycosylation; D020397:Goblet Cells / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D041681:NIH 3T3 Cells; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors
D002241:Carbohydrates; D002704:Chlorates; D004220:Disulfides; C524678:MUC5AC protein, human; C524679:Muc5ac protein, mouse; D055271:Mucin 5AC; D009077:Mucins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D004229:Dithiothreitol; C032706:sodium chlorate