Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region

Anders, David G.; Consigli, R A

doi:10.1128/jvi.48.1.197-205.1983

Cited by 29 publications

(15 citation statements)

References 30 publications

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“…3, lane 2). In vitro surface labeling of polyomavirus virions along with immunological studies have enabled us to suggest that the 29-kDa peptide is cryptic within the virion structure and that the 18-and 16-kDa fragments of VP1, which are exposed on the virion surface, are involved in virus attachment to host cells (1,7,22,25). Calcium binding has previously been shown to be important in maintaining protein conformation (18,28).…”

Section: T-pro La-lys-asn-glu-asn-thr-arg-tyr 240 Phe-gly-asn-tyr-thrmentioning

confidence: 99%

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Localization of calcium on the polyomavirus VP1 capsid protein

Ludlow¹,

Consigli²

1987

J Virol

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Our laboratory has previously shown that the divalent cation Ca2+ is an integral part of the polyomavirus and plays a major role in stabilizing the intact virion structure. In this report, we show that calcium is sequestered on the major capsid protein VP1 of polyomavirus. The virion structural proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose and probed with 45CaC12. Autoradiography revealed 45Ca binding exclusively to VP1. Increasing the amount of VP1 transferred to the nitrocellulose resulted in a concomitant increase in 45Ca binding. 45Ca binding to VP1 could be reduced by competition with an excess of unlabeled CaC12. Separation of the species of VP1 by two-dimensional gel electrophoresis before electroblotting and probing with 45CaCl2 revealed that all six species (A to F) bind the radiolabeled calcium. Formic acid cleavage of the 43-kilodalton (kDa) VP1 protein into 29-, 18-, and 16-kDa fragments before 45Ca-binding analysis revealed that only the 18-and 16-kDa carboxylterminal fragments of this protein bind 45Ca.

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Section: T-pro La-lys-asn-glu-asn-thr-arg-tyr 240 Phe-gly-asn-tyr-thrmentioning

confidence: 99%

“…Localization of the 45Ca-binding domain on the VP1 protein was achieved by first isolating this protein from an SDS-polyacrylamide gel for cleavage into discrete peptides by using formic acid (1). The resulting peptide cleavage products were then separated by SDS-PAGE and electroblotted for use in probing with 45CaC12.…”

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Localization of calcium on the polyomavirus VP1 capsid protein

Ludlow¹,

Consigli²

1987

J Virol

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“…This interest has directed us to investigate in detail the attachment species D, E, and F of the major structural protein VP1, since we have previously shown that these species are responsible for host cell receptor interaction as well as the hemagglutinating ability of the virus (3). Studies have been conducted which indicate that the polyomavirus hemagglutinin and receptor functions reside in, or at least have determinants contained in, the carboxy-terminal 153 amino acids of the VP1 sequence (1). Previous reports have suggested that polyomavirus binding to the host cell surface is a two-step process (3,4,18,21): a nonspecific step involving VP1 species D and F and a specific step involving species E. Polyomavirus agglutination of GPE is believed to be nonspecific, involving species D and F, since both complete virions and empty capsids have this ability yet capsids lack VP1 species E. MKC-py, which exhibited a 10-fold greater ability to agglutinate GPE than that of MEC-py (Table 1), also demonstrated greater nonspecific binding ability to host cells than MEC-py did ( Table 2).…”

Section: Discussionmentioning

confidence: 99%

“…This charge heterogeneity is due at least in part to phosphorylation (1-3, 15, 29, 30), since three of the identified species (D, E, and F) are phosphorylated (2,3). Our laboratory is particularly interested in these three phosphorylated species since we have shown previously that these species correlate with the hemagglutination ability of the virus as well as specific cellular attachment (1,(3)(4)(5).…”

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Differences in biological activity and structural protein VP1 phosphorylation of polyomavirus progeny resulting from infection of primary mouse kidney and primary mouse embryo cell cultures

Ludlow¹,

Consigli²

1987

J Virol

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Both primary mouse kidney and primary mouse embryo cells in culture were used for polyomavirus progeny production. Examination of polyomavirus virion structural integrity revealed that mouse embryo cell progeny contained a threefold greater population of unstable particles when compared with mouse kidney cell progeny. Differences in biological activity between these two progeny virion types were also shown. Mouse kidney cell progeny compared with mouse embryo cell progeny exhibited a 10-fold greater ability to agglutinate guinea pig erythrocytes, a 3-fold lower ability to become internalized into monopinocytotic vesicles, and a 2-fold lower ability to initiate a productive infection based on positive nuclear immunofluorescence when mouse embryo host cell cultures were used. The mouse kidney progeny were also found to bind to host cells less specifically than the mouse embryo cell progeny. When these two progeny virion types were labeled in vivo with 32P and subjected to isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophroesis in the second dimension, differences in the phosphorylation pattern of the major virus-encoded structural protein VP1 species were observed. It was revealed that species D and E of mouse kidney cell progeny were phosphorylated to the same degree, while mouse embryo cell progeny species E and F were phosphorylated equally. These data suggest that the host cells play a role in modulating the biological activity of the virus by affecting the degree and site-specific phosphorylation of the major capsid protein VP1 which may influence the recognition of virus attachment proteins for specific cellular receptors. * Corresponding author. t Contribution no. 86-329-J from the Kansas Agricultural Experiment Station, Kansas State University.

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“…While enormous progress has been made in this field by using de novo designed synthetic peptides (2), the idea of using protein fragments obtained by chemical or enzymatic methods (3) has been only rarely pursued (4). Chemical methods, although equally selective in cleaving proteins on selected residues, are always carried out under harsh conditions that invariably lead to protein unfolding (5). On the other hand, enzymatic proteolysis of folded proteins performed under mild conditions, occurs preferentially on exposed and less structured sites (6,7), and thus can help to isolate shorter polypeptides with partially preserved secondary and tertiary structures.…”

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Protein–Protein Interactions: A Simple Strategy to Identify Binding Sites and Peptide Antagonists

et al. 2009

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Secondary structure motifs and small protein domains can act as building blocks that are isolated and investigated to gain insights into protein global structure but can also modulate interactions with external partners. Most progress has been made in this field using synthetic peptides. Fragmentation of folded proteins by proteolytic enzymes that act preferentially on exposed and less structured sites can help to isolate shorter polypeptides with preserved secondary and tertiary structures that mimic the original protein architecture. Such molecules can be used as probes for structural studies and as tools for in vitro assays to select active fragments useful as agonists or antagonists of the original protein or as scaffolds for the design of more potent and selective ligands. This simple but effective proteolytic methodology has been successfully applied to determine antagonists of protein-protein interactions, allowing the identification of inhibitors with high efficacy and specificity. Here, we present several studies including the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes and phospholipase 1, believed to play a relevant role in the insulin resistance mechanism in phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-overexpressing tissues, the self-association of BCL10 caspase recruitment domain that mediates a protein oligomerization process responsible for NF-kappaB activation and the self-association of growth arrest and DNA damage-inducible factor 45 beta, a major player of the endogenous NF-kappaB-mediated resistance to apoptosis.

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Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region

Cited by 29 publications

References 30 publications

Localization of calcium on the polyomavirus VP1 capsid protein

Localization of calcium on the polyomavirus VP1 capsid protein

Differences in biological activity and structural protein VP1 phosphorylation of polyomavirus progeny resulting from infection of primary mouse kidney and primary mouse embryo cell cultures

Protein–Protein Interactions: A Simple Strategy to Identify Binding Sites and Peptide Antagonists

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