期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：21
页码：11450-11458
DOI：10.1073/pnas.2004034117
出版社：The National Academy of Sciences of the United States of America
摘要：Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments—each containing the respective PEDF-binding region (residues 930 to 938)—are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link–susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner. Pigment epithelium-derived factor (PEDF), which was first discovered in cultured human fetal pigment epithelium and subsequently identified in a wide variety of tissues and organs throughout the body, is classified as a serine protease inhibitor (serpin) superfamily protein ( 1 ). Although to date, PEDF has exhibited no protease inhibitory activity ( 2 ), this secreted protein alternatively exerts multiple important biological functions, including neurotrophic, cell differentiating, antiangiogenic, and antitumor activities ( 1 , 3 ). It is now recognized as a promising therapeutic candidate, particularly for cancer ( 4 ). Multiple cell surface receptors, including the 80-kDa calcium-independent phospholipase A2 (iPLA2)ζ, which belongs to the PLA2/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family, and the nonintegrin 37/67-kDa laminin receptor, have been proposed to interact with this multifunctional protein ( 3 , 4 ). In addition, several binding epitopes of PEDF, including two distinctive receptor-binding regions, a 34-aa segment (Asp44–Asn77) and a 44-aa segment (Val78–Thr121), which exhibit antiangiogenic and proangiogenic/neurotrophic activities, respectively, have been identified and extensively studied ( 5 ). Several reports have suggested that the complexities of PEDF receptor binding are cell context-dependent, and that the existence of several target receptors, even in a single cell phenotype, may lead to contradictory cellular responses (i.e., cell survival or death) depending on the concentration of PEDF as well as the activation state of the target cell in vitro ( 5 ⇓ – 7 ). These findings indicate that PEDF actions are extracellularly regulated in vivo to maintain physiological homeostasis; however, the mechanistic details of this regulation are largely unknown. The crystal structure of human PEDF shows that it adopts a typical serpin fold but has unique asymmetric charge distributions composed of a positively charged surface in its C-terminus region and a negatively charged surface on the opposite side ( 8 ). This surface feature is reflected in the ability of PEDF to bind a variety of extracellular matrix (ECM) components. In particular, the negatively charged region is important for collagen binding ( 9 , 10 ), while the positively charged region is responsible for interactions with glycosaminoglycans, including heparin/heparan sulfate and hyaluronan ( 11 ⇓ – 13 ). As in the case of other serpins and growth factors ( 14 , 15 ), ECM binding is essential for the biological functions of PEDF. Indeed, a cancer cell xenograft experiment using nude mice demonstrated that the collagen-binding null mutant of PEDF completely lost its antiangiogenic and antitumor properties, although the mutant retained binding ability to its target receptors ( 16 ). Using site-specific photocrosslinking coupled with matrix metalloproteinase-1 (MMP-1) digestion, we found that PEDF selectively binds to the C-terminal 1/4 fragment of type I collagen ( 17 ). The binding site was further narrowed down using a series of synthetic homotrimeric peptides containing portions of the type I collagen sequence rich in lysyl and arginyl residues, with which a crucial PEDF-binding site was unexpectedly identified in the C-terminal–specific amphipathic region (KGHRGFSGL; residues 930 to 938), in the type I collagen α1 chain ( 18 ). This remarkable sequence selectivity in collagen recognition suggests that the PEDF–collagen interaction is not simply dictated by a nonspecific ionic interaction hypothesized to be required for its deposition in the ECM but could have additional functional implications in expressing and regulating the biological activities of PEDF. To further investigate this PEDF–collagen interaction, we designed and synthesized heterotrimeric collagen peptides, consisting of two α1 and one α2 chain segments of type I collagen, and elucidated the atomic details of PEDF interaction with the heterotrimeric type I collagen tripe helix. Using this structural information, we propose the mechanisms by which PEDF selectively recognizes a cryptic site on type I collagen α1 chain containing the crosslink-susceptible Lys930 residue. Molecular docking revealed that the PEDF-binding surface is completely masked by the interaction with a nonhelical N-telopeptide segment of the α1 chain via formation of intermolecular crosslinks in mature type I collagen fibrils. Therefore, this crosslink-coupled regulation of PEDF binding permits spatiotemporal action on specific cells that actively remodel their pericellular type I collagen fibrils. Results Design and Synthesis of Heterotrimeric Collagen Peptides. The exact chain staggering of native type I collagen is still unknown; thus, we designed and synthesized heterotrimeric type I collagen peptides in which all three structural isomers—α2α1α1, α1α2α1, and α1α1α2—were represented ( Fig. 1 A and SI Appendix , Fig. S1 ). Each peptide consisted of sequences corresponding to the conserved PEDF-binding region (residues 930 to 938) ( 18 ) of type I collagen (guest regions) flanked by glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp) triplet repeats (host regions) for stabilization of the triple helical structure ( 19 ). We achieved staggering by one residue through selective Cys pairing at the C-terminal region. The resulting host-guest heterotrimeric collagen peptides with α2 chain segments in the leading, middle, and trailing positions were designated CP211, CP121, and CP112, respectively. These peptides share similar triple-helical stabilities, with melting temperatures ( T m ) of ∼40 °C ( SI Appendix , Fig. S2 ). Sedimentation velocity analytical ultracentrifugation (SV-AUC) and isothermal titration calorimetry (ITC) demonstrated that PEDF interacts with all of these heterotrimeric peptides with a stoichiometry of 1:1; however, the three peptides have different binding affinities depending on the chain registry ( Table 1 and SI Appendix , Figs. S3 and S4 ). CP211 showed higher binding affinity for PEDF compared with the other peptides, comparable to the affinity ( K d = ∼0.13 μM) observed for the interaction between PEDF and native type I collagen ( 10 ).
