Alexander W. Koch

Neuropilin-1 Binds to VEGF121 and Regulates Endothelial Cell Migration and Sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF165. Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF165 binding to NRP1, surprisingly reduces VEGF121-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF121 binds directly to NRP1; however, unlike VEGF165, VEGF121 is not sufficient to bridge the NRP1·VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF165, but not VEGF121 binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.

A dimeric viral SET domain methyltransferase specific to Lys27 of histone H3.

Site-specific lysine methylation of histones by SET domains is a hallmark for epigenetic control of gene transcription in eukaryotic organisms. Here we report that a SET domain protein from Paramecium bursaria chlorella virus can specifically di-methylate Lys27 in histone H3, a modification implicated in gene silencing. The solution structure of the viral SET domain reveals a butterfly-shaped head-to-head symmetric dimer different from other known protein methyltransferases. Each subunit consists of a Greek-key antiparallel β-barrel and a three-stranded open-faced sandwich that mediates the dimer interface. Cofactor S-adenosyl-L-methionine (SAM) binds at the opening of the β-barrel, and amino acids C-terminal to Lys27 in H3 and in the flexible C-terminal tail of the enzyme confer the specificity of this viral histone methyltransferase.

The Minimal Essential Unit for Cadherin-mediated Intercellular Adhesion Comprises Extracellular Domains 1 and 2*

N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the “minimal essential unit” to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.

N-Cadherin Prodomain Processing Regulates Synaptogenesis

Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.

MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model

The primary objective of this study was to utilize MR molecular imaging to compare the 3‐dimensional spatial distribution of Robo4 and αVβ3‐integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16‐F10 melanoma‐bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4‐ or αVβ3‐targeted paramagnetic nanoparticles. The percentage of MR signal‐enhanced voxels throughout the tumor volume was low and increased in animals receiving αVβ3‐ and Robo4‐targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4‐ and αVβ3‐targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with αVβ3‐integrin. Robo4‐ and αVβ3‐targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that αVβ3‐integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but αVβ3‐integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with αVβ3‐integrin expression, may help define tumor character prior to and following cancer therapy.—Boles, K. S., Schmieder, A. H., Koch, A. W., Carano, R. A. D., Wu, Y., Caruthers, S. D., Tong, R. K., Stawicki, S., Hu, G., Scott, M. J., Zhang, H., Reynolds, B. A., Wickline, S. A., and Lanza, G. M. MR angiogenesis imaging with Robo4‐ vs. αVβ3‐targeted nanoparticles in a B16/F10 mouse melanoma model. FASEB J. 24, 4262–4270 (2010). www.fasebj.org

Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration

Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 ± 77, 20 ± 3, 288 ± 86 and 322 ± 82 ng/ml (mean ± standard deviation; n ≥ 10), respectively. Anti-NRP1B, a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1B antibody. In rats and monkeys receiving single injections of anti-NRP1B at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1B, respectively. In addition to the soluble NRP1 isoforms, for the first time, a ~120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by Western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1B treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1B induced membrane NRP1 shedding.

Calcium Binding to Extracellular Matrix Proteins, Functional and Pathological Effects

Calcium concentrations in the extracellular space are 4 to 5 orders of magnitude larger than inside cells. These concentrations are highly controlled by a unique receptor system (Brown et al., 1995); Ward and Ricardi, this book; Brown et al., this book). Free calcium concentration in serum has been determined to be 1.2 mM, however, spatial and time-dependent fluctuations have been detected (for a review, see Maurer et al., 1996). Many different proteins in the extracellular space bind calcium ions and a variety of calcium-binding motifs have been identified in these proteins, e.g. the EF-hand motif, which is common in cytosolic proteins (Maurer et al., 1996). Calcium ions bound to extracellular proteins may serve various functions, but primarily stabilize protein structure which explains tight calcium binding (micromolar or smaller K D values). In addition, sites for weakly bound calcium ions may sense variations in extracellular calcium levels and may be involved in regulation (Maurer et al., 1996); (Koch et al., 1997). This chapter will focus on relevant findings concerning functional and pathological effects of calcium binding to two extracellular matrix proteins (fibrillin-1 and COMP) and the cell adhesion protein E-cadherin. Possible molecular mechanisms for these effects will be discussed as well.

A case study on Sema3E-Fc aggregation and assay-dependent differences in quantitation

BACKGROUND In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein. RESULTS An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay. CONCLUSION Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.