REVIEW ARTICLES The Proteolytic Activation of Vascular Endothelial Growth Factor­C M. Lackner1, C. Schmotz2, M. Jeltsch3,4 1 University of Hamburg, Germany; 2 Research Program for Clinical and Molecular Metabolism, University of Helsinki, Finland; 3 Individualized Drug Therapy Research Program, University of Helsinki, Finland; 4 Wihuri Research Institute, Helsinki, Finland Summary Die proteolytische Aktivierung des Vas­ Aktivität von VEGF­C für verschiedene spe ­ The enzymatic cleavage of the protein kulären Endothelzellwachstumsfaktors­C zi fische Funktionen von unterschiedlichen back bone (proteolysis) is integral to many Pro teinasen regu liert wird. Obwohl VEGF­C biological processes, e.g. for the break­ Zusammenfassung in experimentellen Tierversuchen eine zen­ down of proteins in the digestive system. Enzymatische Schnitte der Polypeptidkette trale Stellung in der Tumorprogression und Specific proteolytic cleavages are also used von Proteinen sind Bestandteil vieler biolo­ ­metastasierung einnimmt, ist die Aus­ to turn on or off the activity of proteins. For gischer Prozesse, so z.B. bei der Zerlegung sagekraft der bisherigen korrelativen Stu­ example, the lymphangiogenic vascular von Proteinen während der Verdauung. dien zur Rolle von VEGF­C bei Tumor er ­ endothelial growth factor­C (VEGF­C) is Gezielte enzymatische Schnitte werden kran kungen des Menschen begrenzt. synthesized as a precursor molecule that auch benutzt, um die Aktivität bestimmter Dieser Umstand ist nicht zuletzt in den must be converted to a mature form by the Proteine ein­ oder auszuschalten. So z.B fehlenden Möglichkeiten begründet, zwis­ enzymatic removal of C­ and N­terminal wird der lymphangiogene Vaskuläre En­ chen der inaktiven und den aktiven For­ pro peptides before it can bind and activate dothelzellwachstumsfaktor­C (VEGF­C) als men zu unterscheiden. its receptors. The constitutive C­terminal Vorläufermolekül synthetisiert, welches Schlüsselwörter: VEGF­C, Lymphangio­ cleavage is mediated by proprotein con­ durch die enzymatische Entfernung von genese, Proteinasen, Proteolyse vertases such as furin. The subsequent ac­ Teilen des Proteins in eine aktive Form tivating cleavage can be mediated by at umgewandelt werden muss, bevor es seine least four different proteases: by plasmin, Rezeptoren binden und aktivieren kann. ADAMTS3, prostate­specific antigen (PSA) Dabei werden C­ und N­terminale Propep­ List of Abbreviations and cathepsin D. Processing by different tide von VEGF­C entfernt. Die konstitutive proteases results in distinct forms of "ma­ C­terminale Spaltung wird durch Propro­ ADAM A Disintegrin­ and Metalloproteinase ture" VEGF­C, that differ in their affinity and teinkonvertasen wie Furin vermittelt. Die their receptor activation potential. This darauffolgende, aktivierende Spaltung ADAMTS3 A Disintegrin­ and Metalloproteinase processing is tightly regulated by the kann durch mindestens vier verschiedene with Thrombospondin Motifs­3 CCBE1 protein. CCBE1 regulates the acti­ Proteasen vermittelt werden: Plasmin, CatD Cathepsin D vating cleavage of VEGF­C by ADAMTS3 ADAMTS3, prostata­spezifisches Antigen and PSA, but not by plasmin. During em­ (PSA) und Cathepsin D. Resultierend aus CCBE1 Collagen­ and Calcium­Binding EGF bryonic development of the lymphatic sys­ der Prozessierung durch unterschiedliche domain­containg protein 1 tem, VEGF­C is activated primarily by the Proteasen entstehen unterschiedliche, ADAMTS3 protease. In contrast, it is be­ “reife” VEGF­C­Formen, die sich in ihrer Affi ­ ELISA Enzyme­Linked Immunosorbent Assay lieved that plasmin is responsible for ni tät und ihrem Rezeptor­Akti vier ungs po­ wound healing lymphangiogenesis and tential unterscheiden. Das CCBE1­Protein ECM Extracellular Matrix PSA for tumor­associated pathological lym­ reguliert die Prozessierung von VEGF­C phangiogenesis. Cathepsin D has also been durch ADAMTS3 und PSA, aber nicht die HS Hennekam Syndrome implicated in tumor lymphangiogenesis. In durch Plasmin. Während des physiologis­ addition, cathepsin D in saliva might acti­ chen Wachstums des Lymphgefäßsystems HSPG Heparansulfate Proteoglykan vate latent VEGF­C upon wound licking, in der Embryonalentwicklung wird VEGF­C there by accelerating wound healing. The hauptsächlich durch die ADAMTS3­Pro­ KLK3 Kallikrein­related Peptidase 3 molecular details of proteolytic activation tease aktiviert. Im Unterschied dazu wird (synonymous with PSA) of VEGF­C are only recently extensively ex­ vermutet, dass Plasmin für die Wund­ plored, and we likely do not know yet all heilung und PSA für die mit Tumorwachs­ MMP­3 Matrix­Metalloproteinase­3 ac ti vating proteases. It appears that the ac­ tum assoziierte, pathologische Lymph an ­ tivity of VEGF­C is regulated for different gio genese verantwortlich ist. Cathepsin D PSA Prostata­Specific Antigen spe cific functions by different proteinases. wurde ebenfalls mit der Tumor­Lymphan­ Al though VEGF­C clearly plays a pivotal giogenese in Verbindung gebracht; da rü ­ TGF­β Transforming Growth Factor­β role for tumor progression and metastasis ber hinaus könnte das im Speichel ent hal ­ in experimental animal studies, the rele­ tene Cathepsin D, durch Wundlecken, la ­ VEGF­A Vascular Endothelial Growth Factor­A, vance of most correlative studies on the ten tes VEGF­C aktivieren und dadurch die often simply referred to as “VEGF” role of VEGF­C in human cancers is quite li ­ Wund heilung beschleunigen. Die moleku­ mi ted until now, also due to the lack of laren Details der proteolytischen Akti vier ­ VEGF­C, ­D Vascular Endothelial Growth Factor­C, ­D meth ods to differentiate between inactive ung von VEGF­C werden erst seit kurzer and active forms. Zeit intensiv erforscht und wahrscheinlich VEGFR VEGF Receptor Key Words: VEGF­C, lymphangiogenesis, sind noch nicht alle aktivierenden Prote a ­ proteinases, proteolysis sen bekannt. Jedoch scheint es, dass die VHD VEGF Homology Domain 88 LymphForsch 23 (2) 2019 REVIEW ARTICLES Proteinases (protein generated from the prec ursor trypsi - cleaving enzymes) no gen by auto catalysis. Proteinases (or pro te ases) are enzymes that cleave pro - Activation of proteins teins by hydrolysing the peptide bonds of the protein Many pro teins are produced as backbone (Figure 1). ey oc - inactive pre cur sors and are activated cur in side (intracellular) and by proteolytic cleavage when their out side (extracellular) of cells, function is required. e proteinases and are es sential for a multi - them selves are also produced as in - tude of cell and body func- active pro-proteinases and must be ac - tions. For example, proteinases ti vated by pro teolytic removal of their process antigens in the course pro peptides. is is of uppermost im - of an im mune reaction for por tance, since the uncon trolled acti - antigen presentation, they vi ty of proteinases would other wise de - break down damaged or un ne - stroy cells and decompose the extra cel - Figure 1 ces sary proteins (e.g. in lyso - lular matrix (ECM). Proteolysis. The hydrolytic cleavage of a peptide bond somes) and they digest food e perhaps best known pro teo ly ti - (orange) of a protein (blue background) into two proteins in the gastro in tes tinal cal ly con trolled processes include fragments (red background). The peptide bonds of the tract. In the stomach, for blood coagu la tion, the limitation of protein backbone are shown as thick lines. The amino example, pepsin is generated blood clotting and its reversal, i.e. the acid side chains are symbolized as green circles. Without from the precursor pepsinogen dis solution of blood clots [1,2]. Many enzymatic catalysis by proteinases this chemical by autoproteolysis at a low pH, bood clotting factors are proteinases, reaction is extremely slow. and in the intestine, trypsin is which in turn acti vate other pro te in a - ses, etc. ("proteolytic cas cade") to cata - lyze the proteolytic con version of so lu - ble fibrinogen into poly merizing fibrin in the final step of the blot clotting cas - cade. Activation of growth factors Many growth factors and also some cyto kines are produced as inactive pre - cur sors, which only become active through pro teo lytic cleavage (“pro ces - sing”). Among the better known growth factors that are activated through proteolytic cleavage are e.g. the Transforming Growth Factor-β (TGF-β)[3], but also the lymph an gio - ge nic growth factors VEGF-C and VEGF-D. Many studies have analyzed the me cha nisms and the regulation of Figure 2 VEGF re cep tor activation by VEGFs The growth factors VEGF-A, VEGF-C and VEGF-D and their receptors. The growth and [4], whereas rela tive ly little is known function of blood and lymphatic vessels is controlled by Vascular Endothelial Growth Factors about the upstream pro cesses of mo bi - (VEGFs). VEGF­A is the quintessential growth factor for blood vessels, while VEGF­C is the li sation and activation of VEGFs. quintessential growth factor for lymphatic vessels. VEGF­A is recognised by VEGF receptor­1 (VEGFR­ 1) and VEGF receptor­2 (VEGFR­2). VEGF­C and VEGF­D are recognised by VEGF receptor­3 (VEGFR­3) and, under certain circumstances, also by VEGFR­2. VEGFR­1 is largely specific for endothelial cells of The VEGF family blood vessels and VEGFR­3 for endothelial cells of lymphatic vessels. In contrast, VEGFR­2 is found on e biology of the growth factors both vessel types. If, for example, active VEGF­C or VEGF­D binds to VEGFR­3 on the lymphatic VEGF-C and VEGF-D has been de - endothelial cell surface, the signal is transduced into the cell nucleus, where it provokes a scribed in detail in a previous review proliferative and migratory response, thus initiating vessel growth. LymphForsch 23 (2) 2019 89 REVIEW ARTICLES The C-terminal domain of VEGF-C Similar to VEGF-A, VEGF-C and VEGF-D are also im mo bi lized on cell sur faces and the ECM via their C-ter - minal domain [9]. In contrast to all other VEGF family members, the C- ter minal domain of VEGF-C and VEGF-D blocks the growth factor acti - vity [10]. Most likely, this do main ste - ri cally hinders access to the re cep tor bin ding site.