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J. Biol. Chem., Vol. 281, Issue 8, 4844-4855, February 24, 2006

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Mucin Granule Intraluminal Organization in Living Mucous/Goblet Cells

ROLES OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS AND SECRETION^*

Juan Perez-Vilar¹, Raean Mabolo, Cheryl T. McVaugh, Carolyn R. Bertozzi^¶||**, and Richard C. Boucher

From the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248, the Departments of Chemistry and ^¶Molecular and Cell Biology, University of California, Berkeley, California 94720, and the ^||Howard Hughes Medical Institute and the ^**Material Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460

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), 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 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.

Received for publication, September 26, 2005 , and in revised form, December 22, 2005.

This manuscript is dedicated to the memory of Dr. A. Paradiso, University of North Carolina at Chapel Hill.

^* This work was supported by Cystic Fibrosis Foundation Grants PEREZ3I0 and PEREZV04G0 (to J. P.-V.), National Institutes of Health Grants DK63030 (to J. P.-V.) and GM66047 (to C. R. B.), and the Prostate Cancer Development Research Program Prostate SPORE Award P50 CA89520 (to C. R. B.). Parts of this work were presented at the 2004 and 2005 Meetings of the Biophysical Society in Baltimore, Maryland, and Long Beach, California, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The online version of this article (available at http://www.jbc.org) contains supplemental Figs. s1–s3.

¹ To whom correspondence should be addressed: Cystic Fibrosis/Pulmonary Research and Treatment Center, CB7248, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248. Tel.: 919-843-5142; Fax: 919-966-7524; E-mail: juan_vilar{at}med.unc.edu.

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