Protein-disulfide Isomerase (PDI) in FRTL5 Cells

Abstract

Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser 789 -Met 1172 TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an "escort" for immature TG in acidic post-endoplasmic reticulum compartments is discussed.The functional unit of the thyroid gland is the follicle, which is composed of a monolayer of epithelial cells, the thyrocytes, surrounding a closed space, the follicular lumen. The thyroid prohormone, thyroglobulin (TG), 1 is synthesized by thyrocytes and then secreted into the follicular lumen in which the thyroid hormones themselves are synthesized. Hormonal secretion requires the endocytosis of mature TG (i.e. TG bearing hormone residues), its targeting to lysosomes, and the subsequent release of hormones. Each step of this bidirectional trafficking of TG is subject to regulation, in some cases mediated by molecular chaperones (1-3).Thyrocytes synthesize large amounts of TG (up to 50% of protein synthesis in the gland; Ref. 4). Glycosylation and chaperone-assisted folding of TG monomers (M r 330,000) take place in the endoplasmic reticulum (ER), and involve several chaperones including calnexin, BiP, ERp72, grp94, and grp170 (5-9). The dimerization of monomers, a process thought to abolish the interactions between secreted molecules and chaperones, probably determines the sorting and targeting post-ER of glycoproteins (10). The transit through the Golgi apparatus of newly synthesized TG leads to the modification of some high mannose type N-glycans into N-acetyllactosamine chains. Secreted glycoproteins have an almost unique property, mediated by an unknown regulatory mechanism, in that some of these lactosamine chains of TG are incompletely processed and have accessible GlcNAc moieties (11,12). TG molecules are then targeted to small secretory vesicles and released via the regulated secretory pathway of thyrocytes (13,14).In the follicular lumen, the prohormone ...

Identification of ERp29, an Endoplasmic Reticulum Lumenal Protein, as a New Member of the Thyroglobulin Folding Complex

“…Despite the existence of such retrieval mechanism, many lumenal proteins, such as GRP94, PDI, calreticulin, BiP, and PDI, have been found in the cell exterior (22)(23)(24)(25), and several reports have proposed possible functional significance of such localization (25,32). The instances of the unusual localization of PDI are of particular interest for this study.…”

“…log of PDI despite the absence of the active site motif, and because PDI may function also as a general chaperone, it was proposed that these two proteins might have some overlapping functions (9). However, extracellular function of PDI may differ from the ERp29 role, because PDI was found on the surface of FRTL-5 cells and proposed to react with Tg under acidic conditions (25), whereas our studies in living cells failed to reveal any cell surface expression of ERp29 (results not shown).…”

“…25 and references therein) are found in the culture media of the different primary and transformed cell lines. We examined media collected from the quiescent and hormone-stimulated cells for the presence of ERp29.…”

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

“…The beads were washed with PBS, and bound proteins eluted with SDS sample buffer, resolved on 10% SDS-PAGE, and transferred to nitrocellulose membrane. Anti-PDI polyclonal antibodies (SPA-890, 1/1000) and anti-rabbit antibodies coupled to peroxidase (1/2000, Sigma) were used for staining (15).…”

“…It catalyzes reduction, oxidation, and thiol-disulfide interchange reactions and has important roles in the folding of secretory proteins in the biosynthetic pathway (11,12). On the surface of the cell, it has been shown to cause structural modifications of exofacial proteins involved in biological process (11,(13)(14)(15). Clustered on the lymphocyte surface in the vicinity of CD4-enriched regions (9), PDI may influence the conformation modifications that occur during the interaction of HIV Env with the target cell surface receptors through a partial reorganization of the network of the disulfide bonds of the viral protein.…”