NTR Domain Research Articles

Mono- and Bi-specific Nanobodies Targeting the CUB Domains of PCPE-1 Reduce the Proteolytic Processing of Fibrillar Procollagens

The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies.Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.

De novo structure prediction of meteorin and meteorin-like protein for identification of domains, functional receptor binding regions, and their high-risk missense variants

Meteorin (Metrn) and Meteorin-like (Metrnl) are homologous secreted proteins involved in neural development and metabolic regulation. In this study, we have performed de novo structure prediction and analysis of both Metrn and Metrnl using Alphafold2 (AF2) and RoseTTAfold (RF). Based on the domain and structural homology analysis of the predicted structures, we have identified that these proteins are composed of two functional domains, a CUB domain and an NTR domain, connected by a hinge/loop region. We have identified the receptor binding regions of Metrn and Metrnl using the machine-learning tools ScanNet and Masif. These were further validated by docking Metrnl with its reported KIT receptor, thus establishing the role of each domain in the receptor interaction. Also, we have studied the effect of non-synonymous SNPs on the structure and function of these proteins using an array of bioinformatics tools and selected 16 missense variants in Metrn and 10 in Metrnl that can affect the protein stability. This is the first study to comprehensively characterize the functional domains of Metrn and Metrnl at their structural level and identify the functional domains, and protein binding regions. This study also highlights the interaction mechanism of the KIT receptor and Metrnl. The predicted deleterious SNPs will allow further understanding of the role of these variants in modulating the plasma levels of these proteins in disease conditions such as diabetes. Communicated by Ramaswamy H. Sarma

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Echinoderms: Structure and Possible Functions.

Echinoderms are one of the most ancient groups of invertebrates. The study of their genomes has made it possible to conclude that these animals have a wide variety of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The phylogenetic analysis shows that the MMPs and TIMPs underwent repeated duplication and active divergence after the separation of Ambulacraria (Echinodermata+Hemichordata) from the Chordata. In this regard the homology of the proteinases and their inhibitors between these groups of animals cannot be established. However, the MMPs of echinoderms and vertebrates have a similar domain structure. Echinoderm proteinases can be structurally divided into three groups—archetypal MMPs, matrilysins, and furin-activatable MMPs. Gelatinases homologous to those of vertebrates were not found in genomes of studied species and are probably absent in echinoderms. The MMPs of echinoderms possess lytic activity toward collagen type I and gelatin and play an important role in the mechanisms of development, asexual reproduction and regeneration. Echinoderms have a large number of genes encoding TIMPs and TIMP-like proteins. TIMPs of these animals, with a few exceptions, have a structure typical for this class of proteins. They contain an NTR domain and 10–12 conservatively located cysteine residues. Repeated duplication and divergence of TIMP genes of echinoderms was probably associated with an increase in the functional importance of the proteins encoded by them in the physiology of the animals.

Proteomic profiling reveals biomarkers and pathways in type 2 diabetes risk.

Recent advances in proteomic technologies have made high-throughput profiling of low-abundance proteins in large epidemiological cohorts increasingly feasible. We investigated whether aptamer-based proteomic profiling could identify biomarkers associated with future development of type 2 diabetes (T2DM) beyond known risk factors. We identified dozens of markers with highly significant associations with future T2DM across 2 large longitudinal cohorts (n = 2839) followed for up to 16 years. We leveraged proteomic, metabolomic, genetic, and clinical data from humans to nominate 1 specific candidate to test for potential causal relationships in model systems. Our studies identified functional effects of aminoacylase 1 (ACY1), a top protein association with future T2DM risk, on amino acid metabolism and insulin homeostasis in vitro and in vivo. Furthermore, a loss-of-function variant associated with circulating levels of the biomarker WAP, Kazal, immunoglobulin, Kunitz, and NTR domain–containing protein 2 (WFIKKN2) was, in turn, associated with fasting glucose, hemoglobin A1c, and HOMA-IR measurements in humans. In addition to identifying potentially novel disease markers and pathways in T2DM, we provide publicly available data to be leveraged for insights about gene function and disease pathogenesis in the context of human metabolism.

Native Human Antibody to Shr Promotes Mice Survival After Intraperitoneal Challenge With Invasive Group A Streptococcus.

A vaccine against group A Streptococcus (GAS) has been actively pursued for decades. The surface receptor Shr is vital in GAS heme uptake and provides an effective target for active and passive immunization. Here, we isolated human monoclonal antibodies (mAbs) against Shr and evaluated their efficacy and mechanism. We used a single B-lymphocyte screen to discover the mAbs TRL186 and TRL96. Interactions of the mAbs with whole cells, proteins, and peptides were investigated. Growth assays and cultured phagocytes were used to study the mAbs' impact on heme uptake and bacterial killing. Efficacy was tested in prophylactic and therapeutic vaccination using intraperitoneal mAb administration and GAS challenge. Both TRL186 and TRL96 interact with whole GAS cells, recognizing the NTR and NEAT1 domains of Shr, respectively. Both mAbs promoted killing by phagocytes in vitro, but prophylactic administration of only TRL186 increased mice survival. TRL186 improved survival also in a therapeutic mode. TRL186 but not TRL96 also impeded Shr binding to hemoglobin and GAS growth on hemoglobin iron. Interference with iron acquisition is central for TRL186 efficacy against GAS. This study supports the concept of antibody-based immunotherapy targeting the heme uptake proteins to combat streptococcal infections.

Structural Architecture of the Nucleosome Remodeler ISWI Determined from Cross-Linking, Mass Spectrometry, SAXS, and Modeling

Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, we developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography. The autoinhibiting NegC region does not protrude from the ATPase module as suggested previously. The regulatory NTR domain is located near both ATPase lobes. The full-length enzyme is flexible and can adopt a compact structure in solution with the C-terminal HSS domain packing against the ATPase module. Our data imply a series of conformational changes upon activation of the enzyme and illustrate how the NTR, NegC, and HSS domains contribute to regulation of the ATPase module.

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

The Wnt-signaling pathway is crucial to cell proliferation, differentiation, and migration. The secreted Frizzled-related proteins (sFRPs) represent the largest family of secreted Wnt inhibitors. However, their function in antagonizing Wnt signaling has remained somewhat controversial. Here, we report the crystal structure of Sizzled from Xenopus laevis, the first full-length structure of an sFRP. Tethered by an inter-domain disulfide bond and a linker, the N-terminal cysteine-rich domain (CRD) and the C-terminal netrin-like domain (NTR) of Sizzled are arranged in a tandem fashion, with the NTR domain occluding the groove of CRD for Wnt accessibility. A Dual-Luciferase assay demonstrated that removing the NTR domain and replacing the CRD groove residues His-116 and His-118 with aromatic residues may significantly enhance antagonistic function of Sizzled in inhibiting Wnt3A signaling. Sizzled is a monomer in solution, and Sizzled CRD exhibited different packing in the crystal, suggesting that sFRPs do not have a conserved CRD dimerization mode. Distinct from the canonical NTR domain, the Sizzled NTR adopts a novel α/β folding with two perpendicular helices facing the central mixed β-sheet. The subgroup of human sFRP1/2/5 and Sizzled should have a similar NTR domain that features a highly positively charged region, opposite the NTR-CRD interface, suggesting that the NTR domain in human sFRPs, at least sFRP1/2/5, is unlikely to bind to Wnt but is likely involved in biphasic Wnt signaling modulation. In summary, the Sizzled structure provides the first insights into how the CRD and the NTR domains relate to each other for modulating Wnt-antagonistic function of sFRPs.

Molecular characterization of Netrin-1 and APP receptor binding: New leads to block the progression of senile plaques in Alzheimer's disease

Alzheimer's disease is a growing concern in the context of the increasing lifespan of the populations. The work presented here is part of the fight against this threat. It supports a therapeutic approach to reduce the incidence of Alzheimer's disease, taking advantage of the specific binding of several domains of Netrin-1 to the β-amyloid precursor protein. This basic knowledge shall then be used to predict, design or characterize lead compounds that may in turn inhibit/delay Alzheimer's disease’s progression, extending the therapeutic offer of the other leads already being investigated in this line. The present work is focused on the interaction of the various portions of APP with the three domains of Netrin-1, the so-called LamNT, EGF-like and NTR domains respectively. It reveals in detail which portions of APP and Netrin-1 are specifically involved in these interactions, using ELISA technique in combination with protein-protein binding simulations. So far unsuspected interaction sites located in Netrin-1 EGF-like and NTR domains open possibilities for new therapeutic approaches in which these sites will be specifically targeted.

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19.

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis.

Thioredoxins (TRXs) mediate light-dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox-regulated enzymes. Of the two plastid TRX systems, the ferredoxin-TRX system consists of ferredoxin-thioredoxin reductase (FTR) and multiple TRXs, while the NADPH-dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd-TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO2 fixation rate and lowered non-photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX-regulated enzymes in Calvin-Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose-1,6-bisphosphatase, phosphoribulokinase and CF1 γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC-mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin-TRX system and is crucial when availability of light is limiting photosynthesis.

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in vivo function of reductase and thioredoxin domains

Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.

In the Absence of Thioredoxins, What Are the Reductants for Peroxiredoxins in Thermotoga maritima?

Three peroxiredoxins (Prxs) were identified in Thermotoga maritima, which possesses neither glutathione nor typical thioredoxins: one of the Prx6 class; one 2-Cys PrxBCP; and a unique hybrid protein containing an N-terminal 1-Cys PrxBCP domain fused to a flavin mononucleotide-containing nitroreductase (Ntr) domain. No peroxidase activity was detected for Prx6, whereas both bacterioferritin comigratory proteins (BCPs) were regenerated by a NADH/thioredoxin reductase/glutaredoxin (Grx)-like system, constituting a unique peroxide removal system. Only two of the three Grx-like proteins were able to support peroxidase activity. The inability of TmGrx1 to regenerate oxidized 2-Cys PrxBCP probably results from the thermodynamically unfavorable difference in their disulfide/dithiol E(m) values, -150 and -315 mV, respectively. Mutagenesis of the Prx-Ntr fusion, combined with kinetic and structural analyses, indicated that electrons are not transferred between its two domains. However, their separate activities could function in a complementary manner, with peroxide originating from the chromate reductase activity of the Ntr domain reduced by the Prx domain.

GASP/WFIKKN Proteins: Evolutionary Aspects of Their Functions

Growth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis.

Biological functions of the WAP domain-containing multidomain proteins WFIKKN1 and WFIKKN2

WFIKKN1 and WFIKKN2 are two closely related multidomain proteins consisting of a WAP (whey acidic protein)-, a follistatin-, an immunoglobulin-, two Kunitz-type protease inhibitor-domains and an NTR domain (netrin domain). Recent experiments have shown that both WFIKKN1 and WFIKKN2 bind myostatin and GDF11 (growth and differentiation factor 11) with high affinity and are potent antagonists of these growth factors. Structure-function studies on WFIKKN proteins have revealed that their interactions with GDF8 and GDF11 are mediated primarily by the follistatin and NTR domains.

Expressed sequence tag-simple sequence repeat-based molecular variance in two Salicornia (Amaranthaceae) populations

Salicornia spp is one of the most salt-tolerant vascular plants and is native to salt marshes and estuaries. We developed expressed sequence tag derived-simple sequence repeat (EST-SSR) markers for estimating genetic diversity and marker-assisted Salicornia breeding. Six polymorphic EST-SSRs of 40 detected 27 alleles, ranging from three to five alleles per locus. The average number of alleles per locus was 4.33 and 4.17, and the major allele frequency at locus DY529765 was high, being 0.859 and 0.857 in S. bigelovii and S. europea, respectively. Gene diversity, heterozygosity and polymorphism information content were highest at locus DY529950 and similar in these two species. Gene diversity increased with increase in the number of alleles that had a low major allele frequency at a locus. Six polymorphic loci effectively discriminated 46 taxa into three clusters via different analyses. Significant deviation of F(ST) from zero in three suggested populations for six loci indicated population differentiation and limited gene flow among them. A reduced median network established that taxon SB65 is primitive. SMART (simple modular architecture research tool) analysis of peptide sequences of six EST-SSRs showed that loci DY529765, DY529950 and EC906203 contained transmembrane, TLC, AgrB and NTR domains and might be involved in salinity stress tolerance. These EST-SSRs are a valuable resource for marker development and may be useful in marker-assisted Salicornia breeding.

WFIKKN1 and WFIKKN2 bind growth factors TGFβ1, BMP2 and BMP4 but do not inhibit their signalling activity

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (K(d) ∼ 10(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.

Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.

Strong Cooperativity and Loose Geometry between CUB Domains Are the Basis for Procollagen C-Proteinase Enhancer Activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

Both WFIKKN1 and WFIKKN2 Have High Affinity for Growth and Differentiation Factors 8 and 11

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144-1154). Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold: CONSERVED AND SPECIFIC FEATURES OF THE CUB1 DOMAIN CONTRIBUTE TO THE MOLECULAR BASIS OF PROCOLLAGEN C-PROTEINASE ENHANCER-1 ACTIVITY

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.