Anton-Jan van Zonneveld

Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

Interaction between factor VIII and LDL receptor-related protein. Modulation of coagulation?

Recent reports suggest that the multifunctional receptor low-density lipoprotein receptor-related protein (LRP) may contribute to the regulation of blood coagulation by mechanisms that differ from the simple removal of protease/inhibitor complexes from the circulation. This possibility became apparent from the observation that LRP is involved in down-regulation of Tissue Factor expression at the surface of monocytes and fibroblasts. Furthermore, coagulation Factor VIII and activated Factor IX (Factor IXa) have been identified as proteins that are able to bind to LRP. In the present review, the potential contribution of LRP to the regulation of the coagulation cascade through these novel pathways is discussed, with particular reference to the interaction between LRP and coagulation Factor VIII.

Functional analysis of the human tissue-type plasminogen activator protein: the light chain

A pBR322::Rous sarcoma virus(RSV)-based shuttle vector was used to insert fused genes, composed of the amino-terminal portion of the bacterial chloramphenicol-acetyltransferase gene (cat) and the entire coding region for the C-terminally derived light (L) chain of human tissue-type plasminogen activator (t-PA) cDNA. Cotransfection of rat 3Y1 cells with pRSVneo DNA and pRSVcat/t-PA DNA yielded stably integrated G418-resistant transfectants which contain unrearranged copies of pRSVcat/t-PA DNA. These transfectants synthesize cat/t-PA L-chain mRNA, apparently correctly initiated and terminated. With the help of an enzyme-linked immunosorbent assay (ELISA), it is demonstrated that these cells produce human t-PA antigen. Furthermore, pRSVcat/t-PA L-chain cDNA-containing rat 3Y1 cells synthesize a plasminogen-dependent amidolytic activity which is suppressed by specific anti-human t-PA antibodies. This activity cannot be stimulated by fibrin, a property displayed by native t-PA. It is concluded that the t-PA L-chain cDNA contains the complete genetic information for the plasminogen activator activity.

Small GTP-binding proteins in human endothelial cells

Small GTP‐binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP‐ and GTP‐bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT‐PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP‐binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP‐binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope‐tagged Rab30 cDNA in monkey kidney COS‐1 cells. Immunoelectron‐microscopy of transfected COS‐1 cells indicated that Rab30 is associated with Golgi stacks.

Selection of peptides that bind to plasminogen activator inhibitor 1 (PAI-1) using random peptide phage-display libraries

Large random hexa‐ and decapenta‐peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa‐peptide library on immobilized plasminogen activator inhibitor 1 (PAI‐1) specifically selected a minor fraction of concatemers, indicating that binding to PAI‐1 requires an extended amino acid sequence. Accordingly, the decapenta‐peptide library exclusively yielded PAI‐1 binding peptides of 15 amino acid residues. None of these phage‐bound peptides prevented the interaction between PAI‐1 and its target serine protease urokinase (u‐PA). To isolate peptides that block the interaction between PAI‐1 and u‐PA, phages bound to immobilized PAI‐1 were eluted by incubation with u‐PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI‐1 or u‐PA.