Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.     

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

Strategies for the identification and purification of ligands for orphan biomolecules

Summary The isolation of related genes with evolutionary conserved motifs by the application of polymerase chain reaction-based molecular biology techniques, or from database searching strategies, has facilitated the identification of new members of protein families. Many of these protein molecules will be involved in protein-protein interactions (e.g. growth factors, receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellular process. However, the precise biological function and specific binding partners of these novel proteins are frequently unknown, hence they are known as ‘orphan’ molecules. Complementary technologies are required for the identification of the specific ligands or receptors for these and other orphan proteins (e.g., antibodies raised against crude biological extracts or whole cells). We describe herein several alternative strategies for the identification, purification and characterisation of orphan peptide and protein molecules, specifically the synergistic use of micropreparative HPLC and biosensor techniques. These authors made equivalent contributions.     

In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.     

The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.