Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Abstract

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon of VEGF and TKPPR that the for exon VEGF binding to neuropilin-1 to VEGFR-2. the between neuropilin-1 and that of the reported effects of tuftsin Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon of VEGF and TKPPR that the for exon VEGF binding to neuropilin-1 to VEGFR-2. the between neuropilin-1 and that of the reported effects of tuftsin Tuftsin, a peptide with the and as a peptide the of that tuftsin effects on a of and effects that increased and cells the and tuftsin is reported to effects on the nervous of and of and tuftsin of as of and and with for binding to of and the even though with the and greatly that the of a of the of as TKPPR, tuftsin that for tuftsin the binding and of of tuftsin a tuftsin to that tuftsin to and a affinity on of a with a binding for the and the of of tuftsin the and nervous the tuftsin to to unexpectedly found that and endothelial cells tuftsin and that cells the binding for tuftsin is neuropilin-1 a the and nervous and with a of and that of the reported effects of tuftsin and and endothelial endothelial the of vascular endothelial growth factor vascular endothelial growth peptide TKPPR and and endothelial cells and endothelial cells and on as cells and with cells and with and and and of to as a with with of and of a and for of of and the for the of of and of of the for and with TKPPR with with and with to an the a for of an of of and with of for on an with and with with binding and with an with a for a TKPPR of to to as that and of TKPPR peptide to of the binding to and the of with the to and and as to the on a and the with the for of TKPPR competition and the of the the competition and the of binding as the and to the with of peptide is and is as the and as the peptide with and the with the of the the on the with and of TKPPR of and with of with of and the with and the and TKPPR the of TKPPR TKPPR with as with of for and for to and of the an of the with with binding as of with of the with a of to the to and with the and for the with of to the to the cells with and with as binding on the as that the of and of binding and as the binding cells with an the the as the for binding binding to cells as and binding for the of of binding to and binding with the of with with and for and with with to a with the the as that to binding to binding and for and binding with the and an the TKPPR the of binding increased as as as the had the the and and the competition the and the of of of with with for the of tetrameric TKPPR on a a with an and with binding with a and to with and and with an to for binding with a as a to an to of and to vascular for the binding of with TKPPR to to We the as for to with affinity for to endothelial cells binding to and cells had the TKPPR to the We the binding of to endothelial cells and found that TKPPR peptide binding with an of that the binding to a with tuftsin on endothelial of binding not with TKPPR to that endothelial cells endothelial cells of the of endothelial cells the of tuftsin had with that the of tuftsin on endothelial cells that to tuftsin for vascular with the TKPPR on a TKPPR to a of to the the to to endothelial cells with the the TKPPR did not bind the TKPPR binding to endothelial cells as the of TKPPR peptide to inhibit the binding with an of binding to the of and binding to to the binding to on a the binding of TKPPR to endothelial cells a of for binding of TKPPR with that TKPPR a affinity for the tuftsin tetrameric of TKPPR of binding to growth growth a TKPPR and with for binding to neuropilin-1 endothelial cells of tetrameric competition with for binding to competition of binding to tetrameric of and competition with for binding to tetrameric TKPPR and competition with for binding to cells tetrameric TKPPR and of the peptide that TKPPR binding to endothelial cells not the a of TKPPR, that binding to the tuftsin a on the that VEGF a a binding and that it binding to endothelial cells with an of to and as as to and as as binding to and is the of We that the competition with with VEGF binding to the of had that and TKPPR for binding to a We as the binding TKPPR affinity for the TKPPR binding is inhibited the of that not with found that had on TKPPR binding to growth a factor that to not had the TKPPR to neuropilin-1 a for to block binding to We found that binding with an of that is of the binding TKPPR binding to neuropilin-1 binding to an binding inhibited TKPPR peptide and did not with a and found not to bind to with to block binding to neuropilin-1 TKPPR to inhibit binding to of TKPPR tetrameric of TKPPR of with and TKPPR and and tetrameric with for binding to with of and of the with tetrameric TKPPR that the a a the with to to the of for the as to for the that the a the for the and TKPPR had on binding to to of binding to a On tetrameric TKPPR inhibited binding of of the to the for tetrameric TKPPR on of the VEGF on with competition tetrameric TKPPR for binding to on On cells with tetrameric TKPPR had on binding that the TKPPR with a not is a that reported to as VEGF We found that tetrameric TKPPR to block of binding to cells the a of and reported and and cells a We cells for We with that cells and cells and not the on that VEGF binding to the cells with the of tetrameric TKPPR to block binding to of VEGF and of the VEGF of and cells the of and for of autophosphorylation TKPPR and to as for the binding of VEGF to the that VEGF binding to is for that that not bind to the as that that block the binding of to to not the of VEGF as that to endothelial with tetrameric TKPPR an on the of VEGF of the with the VEGF to endothelial cells the of an as We that TKPPR the of VEGFR-2. the of the to had a and a of TKPPR to and binding of the TKPPR to the of a the of of and to the the with a of on the and and for a TKPPR the to is with the for TKPPR and binding to endothelial and binding of tetrameric TKPPR to of TKPPR binding to and an of TKPPR binding to and an of TKPPR binding to and an and did not bind the TKPPR to and and to a the a of the TKPPR that affinity binding of the to TKPPR and not with the of TKPPR to block binding to and the of the to inhibit binding to with binding of TKPPR to a with and that to block TKPPR binding to and did not the directly the that the TKPPR binding the on VEGF that it bind to that TKPPR binding to and cells for binding the to cells not to cells that the cells and that with an of that the and cells the TKPPR to the not that TKPPR a higher affinity for with the binding to cells binding to binding to binding to binding to binding to cells the of TKPPR of of and of for and the of and for found that the tuftsin TKPPR to it binding to neuropilin-1 and that selectively of exon with exon VEGF VEGF VEGFR-2. is with the of neuropilin-1 as a VEGF the of the for a with to tuftsin and exon is a for the it for not a of and binding and a We that the of exon to neuropilin-1 the for exon and to VEGF for binding to VEGFR-2. with a for the of the the and to to even though it a of the of exon with and the effects of that VEGF with neuropilin-1 exon an the for exon not bind to neuropilin-1 We that affinity for neuropilin-1 that is not to the of with neuropilin-1 to the binding to a the of the to is that a of VEGF the for and to inhibit the binding and of to endothelial cells even though the peptide for exon to the of a the tuftsin as tuftsin to of that neuropilin-1 is a tuftsin not the to of the effects of tuftsin to with it is to that the of neuropilin-1 of effects of the system and with neuropilin-1 reported of the as of tuftsin cells and the nervous and with for binding to neuropilin-1 and reported to of the effects of VEGF of for tuftsin and to the of to VEGF to endothelial cells not reported to tuftsin that is with and that inhibit inhibit tuftsin effects to endothelial cells and on it is that the reported of a TKPPR an greatly increased the binding affinity of the TKPPR for the for competition with VEGF for the to for the the for TKPPR binding for TKPPR to for the reported affinity of tuftsin for cells is the the neuropilin-1 a tuftsin affinity for the is that is that tuftsin the and reported that to an on the of and on the of to as as a tuftsin that TKPPR on the of to to endothelial cells and that binding the with a of TKPPR selectively and even to cells of of for as as that neuropilin-1 is a for the peptide on the of a tuftsin to and the tuftsin and that of the reported effects of tuftsin Tuftsin, a peptide with the and as a peptide the of that tuftsin effects on a of and effects that increased and cells the and tuftsin is reported to effects on the nervous of and of and tuftsin of as of and and with for binding to of and the even though with the and greatly that the of a of the of as TKPPR, tuftsin that for tuftsin the binding and of of tuftsin a tuftsin to that tuftsin to and a affinity on of a with a binding for the and the of of tuftsin the and nervous the tuftsin to to We unexpectedly found that and endothelial cells tuftsin and that cells the binding for tuftsin is neuropilin-1 a the and nervous and with a of and that of the reported effects of tuftsin and and endothelial endothelial the of vascular endothelial growth factor vascular endothelial growth peptide TKPPR and and endothelial cells and endothelial cells and on as cells and with cells and with and and and of to as a with with of and of a and for of of and the for the of of and of of the for and with TKPPR with with and with to an the a for of an of of and with of for on an with and with with binding and with an with a for a TKPPR of to to as that and of TKPPR peptide to of the binding to and the of with the to and and as to the on a and the with the for of TKPPR competition and the of the the competition and the of binding as the and to the with of peptide is and is as the and as the peptide with and the with the of the the on the with and of TKPPR of and with of with of and the with and the and TKPPR the of TKPPR TKPPR with as with of for and for to and of the an of the with with binding as of with of the with a of to the to and with the and for the with of to the to the cells with and with as binding on the as that the of and of binding and as the binding cells with an the the as the for binding binding to cells as and binding for the of of binding to and binding with the of with with and for and with with to a with the the as that to binding to binding and for and binding with the and an the TKPPR the of binding increased as as as the had the the and and the competition the and the of of of with with for the of tetrameric TKPPR on a a with an and with binding with a and to with and and with an to for binding with a as and and endothelial endothelial the of vascular endothelial growth factor vascular endothelial growth peptide TKPPR and and endothelial cells and endothelial cells and on as cells and with cells and with and and and of to as a with with of and of a and for of of and the for the of of and of of the for and with TKPPR with with and with to an the a for of an of of and with of for on an with and with with binding and with an with a for a TKPPR of to to as that and of TKPPR peptide to of the binding to and the of with the to and and as to the on a and the with the for of TKPPR competition and the of the the competition and the of binding as the and to the with of peptide is and is as the and as the peptide with and the with the of the the on the with and of TKPPR of and with of with of and the with and the and TKPPR the of TKPPR TKPPR with as with of for and for to and of the an of the with with binding as of with of the with a of to the to and with the and for the with of to the to the cells with and with as binding on the as that the of and of binding and as the binding cells with an the the as the for binding binding to cells as and binding for the of of binding to and binding with the of with with and for and with with to a with the the as that to binding to binding and for and binding with the and an the TKPPR the of binding increased as as as the had the the and and the competition the and the of of of with with for the of tetrameric TKPPR on a a with an and with binding with a and to with and and with an to for binding with a as a to an to of and to vascular for the binding of with TKPPR to to We the as for to with affinity for to endothelial cells binding to and cells had the TKPPR to the We the binding of to endothelial cells and found that TKPPR peptide binding with an of that the binding to a with tuftsin on endothelial of binding not with TKPPR to that endothelial cells endothelial cells of the of endothelial cells the of tuftsin had with that the of tuftsin on endothelial cells that to tuftsin for vascular with the TKPPR on a TKPPR to a of to the the to to endothelial cells with the the TKPPR did not bind the TKPPR binding to endothelial cells as the of TKPPR peptide to inhibit the binding with an of binding to the of and binding to to the binding to on a the binding of TKPPR to endothelial cells a of for binding of TKPPR with that TKPPR a affinity for the tuftsin tetrameric of TKPPR of binding to growth growth a of the peptide that TKPPR binding to endothelial cells not the a of TKPPR, that binding to the tuftsin a on the that VEGF a a binding and that it binding to endothelial cells with an of to and as as to and as as binding to and is the of We that the competition with with VEGF binding to the of had that and TKPPR for binding to a We as the binding TKPPR affinity for the TKPPR binding is inhibited the of that not with found that had on TKPPR binding to growth a factor that to not had the TKPPR to neuropilin-1 a for to block binding to We found that binding with an of that is of the binding TKPPR binding to neuropilin-1 binding to an binding inhibited TKPPR peptide and did not with a and found not to bind to with to block binding to neuropilin-1 TKPPR to inhibit binding to of TKPPR tetrameric of TKPPR of with and TKPPR and and tetrameric with for binding to with of and of the with tetrameric TKPPR that the a a the with to to the of for the as to for the that the a the for the and TKPPR had on binding to to of binding to a On tetrameric TKPPR inhibited binding of of the to the for tetrameric TKPPR on of the VEGF on with competition tetrameric TKPPR for binding to on On cells with tetrameric TKPPR had on binding that the TKPPR with a not is a that reported to as VEGF We found that tetrameric TKPPR to block of binding to cells the a of and reported and and cells a We cells for We with that cells and cells and not the on that VEGF binding to the cells with the of tetrameric TKPPR to block binding to of VEGF and of the VEGF of and cells the of and for of autophosphorylation TKPPR and to as for the binding of VEGF to the that VEGF binding to is for that that not bind to the as that that block the binding of to to not the of VEGF as that to endothelial with tetrameric TKPPR an on the of VEGF of the with the VEGF to endothelial cells the of an as We that TKPPR the of VEGFR-2. the of the to had a and a of TKPPR to and binding of the TKPPR to the of a the of of and to the the with a of on the and and for a TKPPR the to is with the for TKPPR and binding to endothelial and binding of tetrameric TKPPR to of TKPPR binding to and an of TKPPR binding to and an of TKPPR binding to and an and did not bind the TKPPR to and and to a the a of the TKPPR that affinity binding of the to TKPPR and not with the of TKPPR to block binding to and the of the to inhibit binding to with binding of TKPPR to a with and that to block TKPPR binding to and did not the directly the that the TKPPR binding the on VEGF that it bind to that TKPPR binding to and cells for binding the to cells not to cells that the cells and that with an of that the and cells the TKPPR to the not that TKPPR a higher affinity for with the binding to cells binding to binding to binding to binding to binding to cells the of TKPPR of of and of for and the of and for a to an to of and to vascular for the binding of with TKPPR to to We the as for to with affinity for to endothelial cells binding to and cells had the TKPPR to the We the binding of to endothelial cells and found that TKPPR peptide binding with an of that the binding to a with tuftsin on endothelial of binding not with TKPPR to that endothelial cells endothelial cells of the of endothelial cells the of tuftsin had with that the of tuftsin on endothelial cells that to tuftsin for vascular with the TKPPR on a TKPPR to a of to the the to to endothelial cells with the the TKPPR did not bind the TKPPR binding to endothelial cells as the of TKPPR peptide to inhibit the binding with an of binding to the of and binding to to the binding to on a the binding of TKPPR to endothelial cells a of for binding of TKPPR with that TKPPR a affinity for the tuftsin tetrameric of TKPPR of the peptide that TKPPR binding to endothelial cells not the a of TKPPR, that binding to the tuftsin a on the that VEGF a a binding and that it binding to endothelial cells with an of to and as as to and as as binding to and is the of We that the competition with with VEGF binding to the of had that and TKPPR for binding to a We as the binding TKPPR affinity for the TKPPR binding is inhibited the of that not with found that had on TKPPR binding to growth a factor that to not had the TKPPR to neuropilin-1 a for to block binding to We found that binding with an of that is of the binding TKPPR binding to neuropilin-1 binding to an binding inhibited TKPPR peptide and did not with a and found not to bind to with TKPPR to block binding to neuropilin-1 TKPPR to inhibit binding to of TKPPR tetrameric of TKPPR of with and TKPPR and and tetrameric with for binding to with of and of the with tetrameric TKPPR that the a a the with to to the of for the as to for the that the a the for the and TKPPR had on binding to to On tetrameric TKPPR inhibited binding of of the to the for tetrameric TKPPR on of the VEGF on with competition tetrameric TKPPR for binding to on On cells with tetrameric TKPPR had on binding that the TKPPR with a not VEGFR-2. is a that reported to as VEGF We found that tetrameric TKPPR to block of binding to cells the a of and reported and and cells a We cells for We with that cells and cells and not the on that VEGF binding to the cells with the of tetrameric TKPPR to block binding to and to as for the binding of VEGF to the that VEGF binding to is for that that not bind to the as that that block the binding of to to not the of VEGF as that to endothelial with tetrameric TKPPR an on the of VEGF of the with the VEGF to endothelial cells the of an as We that TKPPR the of VEGFR-2. the of the to had a and a of TKPPR to and binding of the TKPPR to the of a the of of and to the the with a of on the and and for a TKPPR the to is with the for TKPPR and binding to endothelial and did not bind the TKPPR to and and to a the a of the TKPPR that affinity binding of the to TKPPR and not with the of TKPPR to block binding to and the of the to inhibit binding to with binding of TKPPR to a with and that to block TKPPR binding to and did not the directly the that the TKPPR binding the on VEGF that it bind to that TKPPR binding to and cells for binding the to cells not to cells that the cells and that with an of that the and cells the TKPPR to the not that TKPPR a higher affinity for with the found that the tuftsin TKPPR to it binding to neuropilin-1 and that selectively of exon with exon VEGF VEGF VEGFR-2. is with the of neuropilin-1 as a VEGF the of the for a with to tuftsin and exon is a for the it for not a of and binding and a We that the of exon to neuropilin-1 the for exon and to VEGF for binding to VEGFR-2. with a for the of the the and to to even though it a of the of exon with and the effects of that VEGF with neuropilin-1 exon an the for exon not bind to neuropilin-1 We that affinity for neuropilin-1 that is not to the of with neuropilin-1 to the binding to a the of the to is that a of VEGF the for and to inhibit the binding and of to endothelial cells even though the peptide for exon to the of a the tuftsin as tuftsin to of that neuropilin-1 is a tuftsin not the to of the effects of tuftsin to with it is to that the of neuropilin-1 of effects of the system and with neuropilin-1 reported of the as of tuftsin cells and the nervous and with for binding to neuropilin-1 and reported to of the effects of VEGF of for tuftsin and to the of to VEGF to endothelial cells not reported to tuftsin that is with and that inhibit inhibit tuftsin effects to endothelial cells and on it is that the reported of a TKPPR an greatly increased the binding affinity of the TKPPR for the for competition with VEGF for the to for the the for TKPPR binding for TKPPR to for the reported affinity of tuftsin for cells is the the neuropilin-1 a tuftsin affinity for the is that is that tuftsin the and reported that to an on the of and on the of to as as a tuftsin that TKPPR on the of to to endothelial cells and that binding the with a of TKPPR selectively and even to cells of of for as as that neuropilin-1 is a for the peptide on the of a tuftsin to and the tuftsin and that of the reported effects of tuftsin We found that the tuftsin TKPPR to it binding to neuropilin-1 and that selectively of exon with exon VEGF VEGF VEGFR-2. is with the of neuropilin-1 as a VEGF the of the for a with to tuftsin and exon is a for the it for not a of and binding and a We that the of exon to neuropilin-1 the for exon and to VEGF for binding to VEGFR-2. with a for the of the the and to to even though it a of the of exon with and the effects of that VEGF with neuropilin-1 exon an the for exon not bind to neuropilin-1 We that affinity for neuropilin-1 that is not to the of with neuropilin-1 to the binding to a the of the to is that a of VEGF the for and to inhibit the binding and of to endothelial cells even though the peptide for exon to the of a the tuftsin as tuftsin to of that neuropilin-1 is a tuftsin not the to of the effects of tuftsin to with it is to that the of neuropilin-1 of effects of the system and with neuropilin-1 reported of the as of tuftsin cells and the nervous and with for binding to neuropilin-1 and reported to of the effects of VEGF of for tuftsin and to the of to VEGF to endothelial cells not reported to tuftsin that is with and that inhibit inhibit tuftsin effects to endothelial cells and on it is that the reported of a TKPPR an greatly increased the binding affinity of the TKPPR for the for competition with VEGF for the to for the the for TKPPR binding for TKPPR to for the reported affinity of tuftsin for cells is the the neuropilin-1 a tuftsin affinity for the is that is that tuftsin the and reported that to an on the of and on the of to as as a tuftsin We that TKPPR on the of to to endothelial cells and that binding the with a of TKPPR selectively and even to cells of of for as as that neuropilin-1 is a for the peptide on the of a tuftsin to and the tuftsin and that of the reported effects of tuftsin