Limits...
Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection.

Derebe MG, Zlatkov CM, Gattu S, Ruhn KA, Vaishnava S, Diehl GE, MacMillan JB, Williams NS, Hooper LV - Elife (2014)

Bottom Line: Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear.Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection.Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, United States.

Show MeSH

Related in: MedlinePlus

Additional ligand binding studies on human and mouse SAAs.(A–C) Retinyl acetate (A), β-carotene (B), and retinyl palmitate (C) were titrated into hSAA1, mSAA1, mSAA3, hTfr (negative control) and ApoA1 (negative control), and fluorescence quenching was monitored at 334 nm with excitation at 296 nm. Plots are representative of three independent experiments. The Kds are averages of the values derived from the three experiments. (D) Competitive inhibition of retinol binding by cholesterol was quantified. Saturating concentrations of retinol were added to hSAA1, mSAA1, and mSAA3, and fluorescence quenching was monitored as in (A–C). 10 μM cholesterol was added into the assay and inhibition of fluorescence quenching by retinol was monitored. Values are the average ± SEM of triplicate experiments.DOI:http://dx.doi.org/10.7554/eLife.03206.009
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4129439&req=5

fig2s2: Additional ligand binding studies on human and mouse SAAs.(A–C) Retinyl acetate (A), β-carotene (B), and retinyl palmitate (C) were titrated into hSAA1, mSAA1, mSAA3, hTfr (negative control) and ApoA1 (negative control), and fluorescence quenching was monitored at 334 nm with excitation at 296 nm. Plots are representative of three independent experiments. The Kds are averages of the values derived from the three experiments. (D) Competitive inhibition of retinol binding by cholesterol was quantified. Saturating concentrations of retinol were added to hSAA1, mSAA1, and mSAA3, and fluorescence quenching was monitored as in (A–C). 10 μM cholesterol was added into the assay and inhibition of fluorescence quenching by retinol was monitored. Values are the average ± SEM of triplicate experiments.DOI:http://dx.doi.org/10.7554/eLife.03206.009

Mentions: Retinoic acid lacks intrinsic fluorescence but can quench inherent protein fluorescence due to energy transfer from tryptophan residues (Cogan et al., 1976). We therefore measured retinoic acid binding using a modified fluorescence assay that monitored quenching of protein fluorescence (Figure 2C). Titration of all-trans retinoic acid yielded Kds of 268 and 224 nM for retinoic acid binding to hSAA1 and mSAA3, respectively (Figure 2D,E), which are similar to binding affinities calculated for human RBP binding to retinoic acid (Cogan et al., 1976) (Figure 2—figure supplement 1). There was weak binding of retinoic acid to mSAA1 and we were unable to calculate a Kd for the interaction (Figure 2D,E). Thus, while hSAA1 and mSAA3 bind both retinol and retinoic acid, mSAA1 selectively binds retinol. mSAA1 also showed weak binding to other retinoids, including β-carotene and retinyl acetate, while hSAA1 bound these compounds with Kds of 497 and 347 nM, respectively, and mSAA3 bound β-carotene with a Kd of 159 nM (Figure 2—figure supplement 2A,B). All SAA isoforms bound weakly to retinyl palmitate (Figure 2—figure supplement 2C). Since long chain retinyl esters (such as retinyl palmitate) are the major form of stored retinoid in the liver (Vogel et al., 1999), this suggests that SAAs do not transport retinoids for storage. Although a role for SAAs in cholesterol transport and metabolism has been proposed (van der Westhuyzen et al., 2005), we found that cholesterol was unable to competitively inhibit retinol binding to SAAs (Figure 2—figure supplement 2D).


Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection.

Derebe MG, Zlatkov CM, Gattu S, Ruhn KA, Vaishnava S, Diehl GE, MacMillan JB, Williams NS, Hooper LV - Elife (2014)

Additional ligand binding studies on human and mouse SAAs.(A–C) Retinyl acetate (A), β-carotene (B), and retinyl palmitate (C) were titrated into hSAA1, mSAA1, mSAA3, hTfr (negative control) and ApoA1 (negative control), and fluorescence quenching was monitored at 334 nm with excitation at 296 nm. Plots are representative of three independent experiments. The Kds are averages of the values derived from the three experiments. (D) Competitive inhibition of retinol binding by cholesterol was quantified. Saturating concentrations of retinol were added to hSAA1, mSAA1, and mSAA3, and fluorescence quenching was monitored as in (A–C). 10 μM cholesterol was added into the assay and inhibition of fluorescence quenching by retinol was monitored. Values are the average ± SEM of triplicate experiments.DOI:http://dx.doi.org/10.7554/eLife.03206.009
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129439&req=5

fig2s2: Additional ligand binding studies on human and mouse SAAs.(A–C) Retinyl acetate (A), β-carotene (B), and retinyl palmitate (C) were titrated into hSAA1, mSAA1, mSAA3, hTfr (negative control) and ApoA1 (negative control), and fluorescence quenching was monitored at 334 nm with excitation at 296 nm. Plots are representative of three independent experiments. The Kds are averages of the values derived from the three experiments. (D) Competitive inhibition of retinol binding by cholesterol was quantified. Saturating concentrations of retinol were added to hSAA1, mSAA1, and mSAA3, and fluorescence quenching was monitored as in (A–C). 10 μM cholesterol was added into the assay and inhibition of fluorescence quenching by retinol was monitored. Values are the average ± SEM of triplicate experiments.DOI:http://dx.doi.org/10.7554/eLife.03206.009
Mentions: Retinoic acid lacks intrinsic fluorescence but can quench inherent protein fluorescence due to energy transfer from tryptophan residues (Cogan et al., 1976). We therefore measured retinoic acid binding using a modified fluorescence assay that monitored quenching of protein fluorescence (Figure 2C). Titration of all-trans retinoic acid yielded Kds of 268 and 224 nM for retinoic acid binding to hSAA1 and mSAA3, respectively (Figure 2D,E), which are similar to binding affinities calculated for human RBP binding to retinoic acid (Cogan et al., 1976) (Figure 2—figure supplement 1). There was weak binding of retinoic acid to mSAA1 and we were unable to calculate a Kd for the interaction (Figure 2D,E). Thus, while hSAA1 and mSAA3 bind both retinol and retinoic acid, mSAA1 selectively binds retinol. mSAA1 also showed weak binding to other retinoids, including β-carotene and retinyl acetate, while hSAA1 bound these compounds with Kds of 497 and 347 nM, respectively, and mSAA3 bound β-carotene with a Kd of 159 nM (Figure 2—figure supplement 2A,B). All SAA isoforms bound weakly to retinyl palmitate (Figure 2—figure supplement 2C). Since long chain retinyl esters (such as retinyl palmitate) are the major form of stored retinoid in the liver (Vogel et al., 1999), this suggests that SAAs do not transport retinoids for storage. Although a role for SAAs in cholesterol transport and metabolism has been proposed (van der Westhuyzen et al., 2005), we found that cholesterol was unable to competitively inhibit retinol binding to SAAs (Figure 2—figure supplement 2D).

Bottom Line: Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear.Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection.Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, United States.

Show MeSH
Related in: MedlinePlus