| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1,1
* Department of Genitourinary Medical Oncology,
Department of Cancer Biology, and
Department of Biostatistics, The University of Texas M. D. Anderson Cancer, Houston, Texas, USA
1Correspondence: 1515 Holcombe Blvd., Houston, TX 77030-4095, USA. E-mail: watap{at}mdanderson.ord; rpasqual{at}mdanderson.org
SPECIFIC AIMS
The aim of the study was to streamline the procedure for mapping vascular ligand-receptor pairs in patients with in vivo phage display. Specifically, we tested the approach for simultaneous screening of multiple organs with combinatorial phage display libraries in the mouse model.
PRINCIPAL FINDINGS
1. A platform strategy combining advanced molecular biology, biostatistics, and biochemistry has been established that allows to quickly and efficiently identify vascular ligand-receptor pairs in multiple organs
Selectivity of tissue expression for surface molecules of cells lining the vasculature has been uncovered in disease and during normal development. Systematic profiling of selectively expressed "vascular addresses" has revealed prospective molecular targets that may be used to direct therapies to specific tissues. In vivo phage display is a technology used to reveal organ-specific vascular ligand-receptor systems in animal models and, recently, in patients, and to validate them as potential therapy targets. Because a single biopanning round of a phage library may not always sufficiently enrich for organ-homing peptides, until now, phage display screens have encompassed recovery of phage from the organ of interest in three to four rounds of library selection, utilizing one subject per each round. Thus, systematic characterization of the vasculature by using this "conventional" in vivo phage display screen setup has been rate-limited by the need to separately perform individual multiround screens for motifs homing to each tissue studied. To screen phage libraries in patients more efficiently, we have initiated attempts to isolate tissue-homing peptides for several tissues in a single screen. Here, we integrated a comprehensive strategy to simultaneously screen phage display libraries for peptides homing to any number of tissues without the need for an individual subject for each target tissue.
2. We isolated peptides independently homing to six different mouse organs: pancreas, muscle, bowel, uterus, kidney, and brain
To establish the experimental framework for selection of peptides independently homing to different organs, we performed a synchronous in vivo screen of a phage-displayed cyclic random peptide library on six mouse organs: muscle, bowel, uterus, kidney, pancreas, and brain. Three rounds of library selection were done without the step of whole-body perfusion of the vasculature, which was skipped in order to simulate screening conditions to be used in patients. In each round, peptide-displaying phage were isolated from target organs, amplified, and pooled for the next round of selection. To identify organ-homing motifs, we sequenced the DNA corresponding to peptide inserts from phage clones recovered after each of the three rounds of selection for each of the six organs and analyzed the sequences. Preferential cell binding of specifically homing peptides to differentially expressed receptors results in enrichment, defined by the increased frequency of the peptide recovery in each subsequent round of the screen. Thus, we set out to profile the differential distribution of library-encoded peptides among the six organs, which could be used for the subsequent identification of their targets.
3. Novel statistical algorithms have been integrated into the library screening procedure that enable the identification of peptide motifs enriched throughout the successive rounds of the screening
Based on the premise that three residue motifs (tripeptides) provide a sufficient structure for peptide-protein interactions, we performed a computer-assisted analysis of the 7489 tripeptides contained in each direction within the phage-displayed peptides (2620 from the first round; 2554 from the second round; and 2315 from the third round). First, we evaluated the increase in recovery frequency for individual tripeptides in the three consecutive rounds of selection by using the Bayesian Beta/Binomial model and for each organ found a number of tripeptides progressively enriched, thus suggesting their superior affinity and/or specificity. Next, we assessed the significance of motif representation increase on selection by using the Fisher exact test and identified within the enriched motifs those with terminal frequencies higher than those present in the phage library prior to selection. We adapted the Monte Carlo algorithm to confirm that the statistical cutoff for the Fisher exact test was set appropriately and to demonstrate a progressive accumulation of tripeptides isolated with lower P values from the first to the third round, consistent with enrichment of the corresponding motifs. Finally, we adapted the Fisher exact test to analyze the motifs recovered from the third selection round for specificity of tissue homing by identifying tripeptides that were enriched in one of the six organs, but not in the rest of the organs studied.
4. We implemented a concept of performing a systematic "retro-basic local alignment search tool (BLAST)" analysis of the homing peptide sequences against the prototype biological ligands of the candidate peptide-bound receptors in order to validate the targeted receptors and to map sites of ligands involved in receptor interaction
To identify candidate biological ligands mimicked by homing peptides, we chose an approach based on the previous notion that peptide motifs binding to cell surface receptors often mimic native ligands of these receptors. We used the online ClustalW software to determine extended tripeptide-containing motifs responsible for organ homing and searched a nonredundant database of mouse proteins to identify regions of similarity within proteins potentially mimicked by the motifs using the NCBI BLAST. This revealed 19 motifs as segments of extracellular signaling factors that had been reported to regulate organ-dependent vascular growth or homeostasis and, in some cases, identified several motifs that homed to the same organ and that matched different domains within the same protein. For some of the organs, BLAST identified matches of homing tripeptides to different ligands that share a receptor with a functional role in vascular biology in the target organ, which strongly suggests such a receptor as a vascular zip code.
To demonstrate the possibility of efficient characterization of circulation-accessible receptors by synchronous biopanning, as a proof-of-principle, we chose to validate the PRL receptor (PRLR) as a peptide target in the pancreas. PL-I and PLP-M identified by BLAST analysis to contain pancreas-targeted peptide sequences (ASVL, WSGL, and SWSG) belong to the conserved family of PRL-like peptidic hormones that have been shown to function in the pancreas during pregnancy. Because PRLR is the only known receptor for these proteins, we proposed that the selected peptide motifs target PRLR in vivo by mimicking PRL family hormones. To validate a biochemical interaction of pancreas-homing motifs with PRLR, we screened pancreas-homing phage (pooled clones recovered in rounds 2 and 3) against recombinant PRLR, as well as against PRLR expressed on the surface of COS-1 cells. As a result, we selected seven dominating phage-peptides that bound to PRLR but not to control proteins. Remarkably, computer-assisted "retro-BLAST" analysis of sequences revealed that all of the selected peptides contained amino acid motifs similar to those present in proteins of PRL family. The cluster of matches identified around one of the hormone domains that had been shown to mediate receptor interaction supports our conclusion that the peptides selected mimic the PRLR-binding domain of placental lactogens.
5. To demonstrate the efficiency of the approach, we validated prolactin receptor (PRLR) as a target of pancreas-homing peptides mimicking prolactin-like hormones and as a previously unrecognized vascular marker
As a test case for the biochemical validation of PRLR as a pancreatic vascular marker, we chose CRVASVLPC: the peptide that contained a pancreas-enriched SVL tripeptide and was also recovered as a PRLR-binding prolactin mimic. To demonstrate direct physical interaction between CRVASVLPC and PRLR, we tested binding of CRVASVLPC-phage to COS-1 cells transfected with PRLR and found it to be 9-fold higher than its nonspecific binding to nontransfected COS-1 cells that served as a negative control. The CRVASVLPC motifs bound to PRLR in both forward and reverse orientation in the context of phage, and alanine-scanning mutagenesis demonstrated that all of the residues within the RVASVLP sequence were important for PRLR binding. To further demonstrate the specific affinity of the CRVASVLPC motif for its receptor, we showed that the PRL mimic specifically bound to cells expressing PRLR by using immunofluorescence: phage displaying either CRVASVLPC or CPLVSAVRC were found bound and internalized specifically by cells expressing PRLR, but not by nonexpressing control cells, whereas none of the CRVASVLPC mutants displayed detectable PRLR-expressing cell binding and internalization. Finally, since the SVL tripeptide found within the PRL mimic CRVASVLPC was isolated from the pancreas, we evaluated weather the motif homes to PRLR in the pancreatic blood vessels. We showed that the previously reported pancreatic expression of mouse PRLR protein in the vasculature and in the islets closely resembles the in vivo distribution of phage displaying the CRVASVLPC motif. Taken together, these data indicate that the peptide CRVASVLPC binds to PRLR and suggests that it targets vasculature-exposed PRLR in the pancreas.
CONCLUSIONS AND SIGNIFICANCE
An important aspect of experimental biology is the identification of molecular targets and the design of methods for their functional validation. Here, we tested the approach for synchronous biopanning in mice by selecting homing peptide ligands for six different organs in a single screen and prioritizing them by using software compiled for statistical validation of peptide biodistribution specificity. Both Bayesian and frequentist statistics have led to the identification of largely overlapping populations of peptide sequences enriched in the target organs, thus reinforcing the validity of the identified homing motifs. Based on similarity of the selected peptide motifs to mouse proteins, we identified a number of motif-containing biological candidates for ligands binding to organ-selective receptors. To demonstrate that this methodology can lead to targetable ligand-receptor systems, we validated one of the pancreas-homing peptides as a mimic peptide of natural prolactin receptor ligands. A concept to systematically "retro-BLAST" the receptor-binding peptides onto prototype ligands in order to map sites of ligands involved in receptor interaction allowed us to identify multiple additional PRL mimics. The successful validation of PRLR as a PRL-mimicking peptide target establishes this receptor as a previously unrecognized vascular marker and demonstrates the viability of our approach.
The high-throughput targeting strategy bestowed by the synchronous biopanning reported in this study will open new possibilities for rapid and efficient identification and validation of vascular receptors and their ligand-directed targeting. This approach for screening phage libraries in vivo enables highly efficient isolation of organ-homing ligands from combinatorial libraries and may streamline vascular mapping efforts, thus leading to a better understanding of the functional protein–protein interactions in the vasculature. The synchronous approach to in vivo phage display provides an advantage over the conventional approach because multiple organs internally control for organ selectivity of each other in the successive rounds of selection. Direct combinatorial screenings recently initiated in patients have opened the possibility for systematic isolation of peptide ligands for therapeutic targeting. Synchronous selection of homing peptides for multiple tissues will accelerate the process of phage display-based vascular mapping in vivo and may prove particularly relevant for patient studies, allowing efficient high-throughput selection of targeting ligands for multiple organs in a single screening. In the future, the use of peptide libraries for probing the vasculature in individual patients for diagnostic and therapeutic purposes may become a reality. In summary, the combinatorial methodological platform optimized here for identification of differentially expressed vascular ligand-receptor pairs will greatly advance the initiative in human vasculature mapping.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5186fje
This article has been cited by other articles:
|
|
 |
|
  A. Aartsma-Rus and G.-J. B. van Ommen Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications RNA, October 1, 2007; 13(10): 1609 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A.H. Jarvinen and E. Ruoslahti Molecular Changes in the Vasculature of Injured Tissues Am. J. Pathol., August 1, 2007; 171(2): 702 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. L. T. Ballard, J. M. Holm, and J. M. Edelberg Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling Physiol Genomics, September 14, 2006; 26(3): 202 - 208. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
