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From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

ABSTRACT: Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, alpha and beta, based on their amino acid sequences. Alpha bundlins are also N-acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas beta bundlins do not display these characteristics. The four surface-exposed regions of amino acid sequence heterogeneity between alpha and beta bundlin were therefore investigated as potential LacNAc-specific carbohydrate-binding domains in a bundlin. Mutation of one of these domains, 137-GENNI-141, in alpha(1) bundlin to that of beta bundlin (136-SPDST-140) resulted in BFP that no longer bound to LacNAc or HEp-2 cells. Conversely, mutating the beta3 bundlin gene to encode the alpha bundlin sequence at this domain resulted in the gain of HEp-2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI-->GENNT altered the alpha1 bundlin carbohydrate-binding specificity from LacNAc to the Lewis X glycan sequence.

SUBMITTER: Humphries RM

PROVIDER: S-EPMC2719252 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

Humphries Romney M RM Donnenberg Michael S MS Strecker Jonathan J Kitova Elena E Klassen John S JS Cui Lina L Griener Thomas P TP Mulvey George L GL Armstrong Glen D GD

Molecular microbiology 20090414 4

Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, alpha and beta, based on their amino acid sequences. Alpha bundlins are also N-acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas beta bundlins do not display these character ...[more]

PMID: 19400799

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Project description:BackgroundLong Interspersed Element 1 (LINE-1) is a retrotransposon that is present in 500,000 copies in the human genome. Along with Alu and SVA elements, these three retrotransposons account for more than a third of the human genome sequence. These mobile elements are able to copy themselves within the genome via an RNA intermediate, a process that can promote genome instability. LINE-1 encodes two proteins, ORF1p and ORF2p. Association of ORF1p, ORF2p and a full-length L1 mRNA in a ribonucleoprotein (RNP) particle, L1 RNP, is required for L1 retrotransposition. Previous studies have suggested that fusion of a tag to L1 proteins can interfere with L1 retrotransposition.ResultsUsing antibodies detecting untagged human ORF1p, western blot analysis and manipulation of ORF1 sequence and length, we have identified a set of charged amino acids in the C-terminal region of ORF1p that are important in determining its subcellular localization. Mutation of 7 non-identical lysine residues is sufficient to make the resulting ORF1p to be predominantly cytoplasmic, demonstrating intrinsic redundancy of this requirement. These residues are also necessary for ORF1p to retain its association with KPNA2 nuclear pore protein. We demonstrate that this interaction is significantly reduced by RNase treatment. Using co-IP, we have also determined that human ORF1p associates with all members of the KPNA subfamily.ConclusionsThe prediction of NLS sequences suggested that specific sequences within ORF1p could be responsible for its subcellular localization by interacting with nuclear binding proteins. We have found that multiple charged amino acids in the C-terminus of ORF1p are involved in ORF1 subcellular localization and interaction with KPNA2 nuclear pore protein. Our data demonstrate that different amino acids can be mutated to have the same phenotypic effect on ORF1p subcellular localization, demonstrating that the net number of charged residues or protein structure, rather than their specific location, is important for the ORF1p nuclear localization. We also identified that human ORF1p interacts with all members of the KPNA family of proteins and that multiple KPNA family genes are expressed in human cell lines.

Dataset Information

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

Publications

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

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