Richard S. Johnson

An Essential Role for Ectodomain Shedding in Mammalian Development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

TRAIL‐R2: a novel apoptosis‐mediating receptor for TRAIL

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL‐R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL‐R2, by ligand‐based affinity purification and subsequent molecular cloning. TRAIL‐R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL‐R2 contains two extracellular cysteine‐rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell‐surface‐expressed TRAIL‐R2, and TRAIL‐induced apoptosis is inhibited by a TRAIL‐R2–Fc fusion protein. TRAIL‐R2 mRNA is widely expressed and the gene encoding TRAIL‐R2 is located on human chromosome 8p22‐21. Like TRAIL‐R1, TRAIL‐R2 engages a caspase‐dependent apoptotic pathway but, in contrast to TRAIL‐R1, TRAIL‐R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.

A Poxvirus-Encoded Semaphorin Induces Cytokine Production from Monocytes and Binds to a Novel Cellular Semaphorin Receptor, VESPR

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.

De Novo sequencing and homology searching

In proteomics, de novo sequencing is the process of deriving peptide sequences from tandem mass spectra without the assistance of a sequence database. Such analyses have traditionally been performed manually by human experts, and more recently by computer programs that have been developed because of the need for higher throughput. Although powerful, de novo sequencing often can only determine partially correct sequence tags because of imperfect tandem mass spectra. However, these sequence tags can then be searched in a sequence database to identify the exact or a homologous peptide. Homology searches are particularly useful for the study of organisms whose genomes have not been sequenced. This tutorial will present background important to understanding de novo sequencing, suggestions on how to do this manually, plus descriptions of computer algorithms used to automate this process and to subsequently carryout homology-based database searches. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 1).

A Proteomic Approach for the Identification of Cell-surface Proteins Shed by Metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGFα) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGFα-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides Application to the Study of Cell Surface Proteolysis

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.