Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jun;78(12):6431-8.
doi: 10.1128/JVI.78.12.6431-6438.2004.

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice

Vibhudutta Awasthi  1 , George Meinken, Karen Springer, Suresh C Srivastava, Paul Freimuth
Affiliations

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice

Vibhudutta Awasthi et al. J Virol. 2004 Jun.

Abstract

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Stability and activity of knob trimers. Cleared lysates of E. coli cells expressing wild-type (WT) Ad2 knob were mixed with Laemmli sample buffer and incubated for 5 min at room temperature (−) or in a boiling water bath (+ and Δ) before electrophoresis in an SDS-polyacrylamide gel. Ad2 knob protein in the unheated sample migrated to a position indicated by K3, corresponding to the intact knob trimer (lane 1). After boiling, Ad2 knob protein migrated to a position indicated by K1, corresponding to the dissociated monomeric knob polypeptide (lane 2). Samples of knob domains from wild-type Ad2 (lanes 3 and 4), Ad2 mutants SP/EA (lanes 5 and 6) and C428N (lanes 7 and 8), and wild-type Ad12 (lanes 9 and 10) were analyzed for trimer stability, as described above, after storage of the purified proteins for several days at 4°C. Wild-type Ad2 knob trimers were dissociated to polypeptide monomers and dimers by incubation in Laemmli buffer at room temperature (lane 4), whereas Ad2 SP/EA, Ad2 C428N, and wild-type Ad12 trimers were stable under these conditions (lanes 6, 8, and 9, respectively). Intact knob trimers do not uniformly bind SDS; therefore, the different electrophoretic mobilities of Ad2 and Ad12 knob trimers result from differences in surface charge rather than in molecular size. Proteins were visualized by staining the gels with Coomassie blue. The molecular sizes (in kilodaltons) of protein standards loaded in lanes M are indicated.
FIG. 2.
FIG. 2.
Structural features that impact Ad2 knob trimer stability. (A) Spatial orientation of the cysteine 428 of each polypeptide subunit of the wild-type Ad2 knob trimer. Disulfide bond formation upon air oxidation may produce the Ad2 knob polypeptide dimer species resolved by SDS-polyacrylamide gel electrophoresis (Fig. 1) and render the knob trimer sensitive to denaturation by SDS. Yellow, sulfur atoms. (B) Portion of the surface of the Ad2 knob trimer, showing the juxtaposition of lysine 420 from polypeptide chain A and lysine 513 from chain C. Electrostatic repulsion between these lysines may destabilize the trimer interface and promote denaturation by SDS. Also shown are aspartate 406 and serine 408 from polypeptide chain A. Mutation of serine 408 to glutamic acid renders the knob trimer resistant to denaturation by SDS. The introduced negative charge of Glu408 therefore may balance the positive charge on lysine 420, reducing the electrostatic repulsion across the trimer interface. Both panels were produced with the SwissPDB Viewer program (17).
FIG. 3.
FIG. 3.
(A) 131I-labeled Ad2 knob mutants (C428N and SP/EA) and unlabeled wild-type (WT) Ad2 knob protein were incubated for 5 min at room temperature in buffer alone (lanes 2, 4, and 6, respectively) or in buffer containing the recombinant immunoglobulin variable-type domain of CAR (lanes 3, 5, and 7, respectively) prior to electrophoresis in polyacrylamide gels under nondenaturing conditions to detect the formation of knob-CAR complexes. Samples of CAR D1 alone were loaded in lanes 1 and 8. Proteins were visualized by staining the gel with Coomassie blue. (B) SDS-polyacrylamide gel analysis, as described for Fig. 1, of wild-type and mutant Ad2 knob proteins after labeling with 131I by the iodobead method. Iodination partially destabilized the C428N (lane 4) but not the SP/EA (lane 2) mutant. Proteins were visualized by staining the gel with Coomassie blue.
FIG. 4.
FIG. 4.
Biodistribution of radioiodinated Ad2 knob proteins. (A) Wild-type (WT) and mutant Ad2 knob proteins were directly labeled with 131I by the iodobead method (Pierce Biotechnology, Inc.), and then injected intravenously into mice (via the tail vein). After 6 h of circulation, mice were sacrificed, and the radioactivity in dissected organs was measured. BL, blood; SP, spleen; ST, stomach; LI, liver; KI, kidney; HE, heart; LU, lung; MU, muscle (data for muscles were not collected in the experiment shown). Wild-type and C428N mutant Ad2 knobs have equivalent CAR-binding activities, whereas the SP/EA mutant is unable to bind to CAR. Differences between radioactivity levels in organ samples from mice injected with wild-type versus C428N knob were not significant (P > 0.05). Significant differences (P < 0.05) in radioactivity levels in the blood, liver, kidneys, heart, and lungs were observed between mice injected with the non-CAR-binding SP/EA mutant and the CAR-binding wild-type or C428N knob. (B) The same experiment as that for which results are shown in panel A was performed, except that knob proteins were indirectly labeled with 125I by using the Bolton-Hunter reagent (6). Differences in radioactivity levels in organs from mice injected with SP/EA versus C428N knob were all significant (P < 0.05) except for levels in the stomach.
FIG. 5.
FIG. 5.
Biodistribution of radioiodinated Ad2 C428N protein in the presence of excess unlabeled C428N protein. (A) Ad2 C428N mutant protein was directly iodinated with 131I and injected intravenously into mice (via the tail vein) in the presence (+CC) or absence of a 10-fold excess of coinjected unlabeled C428N protein. After 6 h of circulation, mice were sacrificed, and radioactivity in dissected organs was measured. Organ abbreviations are explained in the legend to Fig. 4. All differences in radioactivity levels in organs between mice injected with 131I-labeled C428N alone and mice coinjected with excess unlabeled C428N were significant (P < 0.05) except for levels in the liver and muscle. (B) Mice were coinjected intravenously with 131I-labeled Ad2 C428N protein and a 10-fold molar excess of unlabeled C428N protein. Mice were sacrificed after 1.5, 6, or 24 h of circulation, and radioactivity in dissected organs was measured. Differences in organ radioactivity levels between the 1.5- and 24-h samples were all significant (P < 0.05). Differences between samples taken at 1.5 versus 6 h were significant (P < 0.05) for all organs except the spleen, lungs, and muscle. Differences between samples taken at 6 versus 24 h were significant for all organs except the lungs.
FIG. 6.
FIG. 6.
Biodistribution of wild-type and mutant Ad12 knob proteins. (A) CAR-binding activities of wild-type (WT) Ad12 knob and the Ad12 knob P417S and PP/EA mutants were measured by fluorescent anisotropy using a fluorescein-labeled CAR probe as described previously (22). (B) Wild-type and mutant Ad12 knob proteins were directly labeled with 131I and injected intravenously into mice (via the tail vein). After 6 h of circulation, mice were sacrificed, and radioactivity in dissected organs was measured. Organ abbreviations are explained in the legend to Fig. 4. Differences in radioactivity levels in organs from mice injected with the CAR-binding wild-type or P417S knob were not significant except for levels in the heart (P < 0.05). Differences between samples from mice injected with the nonbinding PP/EA mutant versus wild-type knob were significant (P < 0.05) except for the spleen, kidneys, and lungs. Differences between PP/EA and P417S mutant samples were all significant (P < 0.05) except for kidney samples.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ashbourne Excoffon, K. J., T. Moninger, and J. Zabner. 2003. The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain. J. Virol. 77:2559-2567. - PMC - PubMed
    1. Bai, M., B. Harfe, and P. Freimuth. 1993. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J. Virol. 67:5198-5205. - PMC - PubMed
    1. Bergelson, J. M. 2003. Virus interactions with mucosal surfaces: alternative receptors, alternative pathways. Curr. Opin. Microbiol. 6:386-391. - PubMed
    1. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-1323. - PubMed
    1. Bewley, M. C., K. Springer, Y. B. Zhang, P. Freimuth, and J. M. Flanagan. 1999. Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR. Science 286:1579-1583. - PubMed

Publication types

Substances

LinkOut - more resources