Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein

  • Richard H. Kimura,

    Affiliation Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

  • Douglas S. Jones,

    Affiliation Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

  • Lei Jiang,

    Affiliation Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

  • Zheng Miao,

    Affiliation Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

  • Zhen Cheng ,

    zcheng@stanford.edu (ZC); jennifer.cochran@stanford.edu (JRC)

    Affiliation Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

  • Jennifer R. Cochran

    zcheng@stanford.edu (ZC); jennifer.cochran@stanford.edu (JRC)

    Affiliation Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America

Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein

  • Richard H. Kimura, 
  • Douglas S. Jones, 
  • Lei Jiang, 
  • Zheng Miao, 
  • Zhen Cheng, 
  • Jennifer R. Cochran
PLOS
x

Abstract

Background

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Methodology/Principal Findings

Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to αvβ3 and αvβ5 integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α5β1 and αiibβ3. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to αvβ3 and αvβ5 integrins. A 64Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.

Conclusions/Significance

We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.

Introduction

Cystine-knot miniproteins, also known as knottins, are small polypeptides (20–60 amino acids) that have an interwoven disulfide-bonded framework, triple-stranded β-sheet fold, and possess one or more solvent exposed loops that mediate binding to diverse targets [1], [2]. Knottin family members, which include toxins, antimicrobials, ion channel inhibitors, and protease inhibitors, share little sequence homology apart from their core cysteine residues [3][5]. As a result, binding epitopes have been introduced into knottin peptides to impart them with new molecular recognition properties without abolishing their three-dimensional fold [6][12]. The Ecballium elaterium trypsin inhibitor (EETI-II) knottin contains three disulfide bonds and binds to and inhibits trypsin through a single 6-amino acid loop [13], [14]. In pivotal studies, EETI-II was used as a molecular scaffold by rationally substituting this trypsin binding loop (PRILMR) with grafted biologically-active peptides against targets such as elastase, thrombopoietin, and integrins [7], [9], [10].

Integrins are a family of α/β heterodimeric adhesion receptors that have distinct ligand binding specificities and cell signaling properties [15]. Non-invasive molecular imaging agents that target tumor-related integrin receptors will play an important role in earlier cancer detection, disease staging, and management [16], [17]. We recently used yeast surface display, a combinatorial method, to identify EETI-II-based knottin peptides that bound with high (low nM) affinity to αvβ3vβ5 or αvβ3vβ55β1 integrins [8], which are overexpressed on tumors or their neovasculature and mediate angiogenesis and metastasis [18][21]. In mouse tumor models, radiolabeled versions of these integrin-binding knottin peptides exhibited high tumor uptake with low background in non-target tissue (i.e. liver and kidney) [22], [23].

While our previous study focused on combinatorial libraries of the EETI-II trypsin binding loop (Loop 1), additional work by our group demonstrated high tolerance of length and sequence diversity in other EETI-II loops [24]. In particular, a loop containing the sequence GPNGF (Loop 3) accommodated broad sequence diversity and tolerated a wide range of loop lengths beyond its original 5 amino acids. In the current study, our goal was to further explore the utility of knottins as molecular scaffolds for polypeptide engineering by evaluating the ability to mutate two structurally-adjacent loops within EETI-II. We used yeast surface display to engineer an EETI-II-based knottin peptide that contains an 11-amino acid sequence in place of Loop 1, and a 10-amino sequence in place of Loop 3. As a model system, each engineered loop contained a separate Arg-Gly-Asp (RGD) integrin-binding motif [25], whose flanking residues were optimized to bind with high affinity to tumor-associated integrin receptors. We measured the binding affinity and specificity of this engineered knottin peptide against cell lines expressing particular integrin receptors. In addition, we radiolabled this knottin peptide and evaluated its ability to target tumors in mouse xenograft models. Importantly, we demonstrated that multiple loops of a knottin peptide, comprising 21 non-native amino acid residues, can be engineered to bind with high affinity to integrin receptors, and the resultant peptide can be used as a probe for non-invasive molecular imaging applications. These findings expand the potential of the cystine-knot scaffold for future studies where multiple knottin loops can be simultaneously engineered to bind to exogenous targets. Such capability will be important, for example, in engineering binders against molecular targets