Andrew J. McKnight

The EGF-TM7 family: unusual structures at the leukocyte surface.

The isolation of cDNA clones encoding mouse F4/80, human epidermal growth factor (EGF) module‐containing mucin‐like hormone receptor 1 (EMR1) and human CD97 has resulted in the description of a novel family of seven‐transmembrane spanning cell surface molecules. These members of the EGF‐TM7 family are characterized by a variable number of NH2‐terminal EGF domains and seven transmembrane‐spanning hydrophobic regions, which demonstrate a degree of sequence homology to the corresponding region in members of the G‐protein‐coupled peptide hormone receptor family. The F4/80 molecule is macrophage‐restricted, as determined by immunohistochemical analysis of a wide range mouse tissues, while mRNA transcripts encoding EMR1, the predicted human F4/80 homologue, also appear to be abundantly expressed by cells of the myelomonocytic lineage. CD97, for which a cellular ligand has been identified (CD55), is expressed on a diverse array of leukocytes and is markedly upregulated on activated T and B cells. The regulation of expression of the EGF‐TM7 genes, the physiologic function(s) of these novel receptors and the further identification of their ligands is the subject of both intense study and this review. J. Leukoc. Biol. 63: 271–280; 1998.

Molecular Analysis of the Epidermal Growth Factor-like Short Consensus Repeat Domain-mediated Protein-Protein Interactions DISSECTION OF THE CD97-CD55 COMPLEX

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 μm) and rapid off-rate (at least 0.6 s−1). The interaction is Ca2+ -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with aK D at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.

Inactivation of the F4/80 Glycoprotein in the Mouse Germ Line

ABSTRACT Macrophages play a crucial role in the defense against pathogens. Distinct macrophage populations can be defined by the expression of restricted cell surface proteins. Resident tissue macrophages, encompassing Kupffer cells of the liver and red pulp macrophages of the spleen, characteristically express the F4/80 molecule, a cell surface glycoprotein related to the seven transmembrane-spanning family of hormone receptors. In this study, gene targeting was used to simultaneously inactivate the F4/80 molecule in the germ line of the mouse and to produce a mouse line that expresses the Cre recombinase under the direct control of the F4/80 promoter (F4/80-Cre knock-in). F4/80-deficient mice are healthy and fertile. Macrophage populations in tissues can develop in the absence of F4/80 expression. Functional analysis revealed that the generation of T-cell-independent B-cell responses and macrophage antimicrobial defense after infection with Listeria monocytogenes are not impaired in the absence of F4/80. Interestingly, tissues of F4/80-deficient mice could not be labeled with anti-BM8, another macrophage subset-specific marker with hitherto undefined molecular antigenic structure. Recombinant expression of a F4/80 cDNA in heterologous cells confirmed this observation, indicating that the targets recognized by the F4/80 and BM8 monoclonal antibodies are identical.

Membrane Molecules as Differentiation Antigens of Murine Macrophages

Publisher Summary The membrane molecules play an important role in all aspects of macrophage immunobiology. Some of the roles played by them are as receptors for growth and differentiation factors, as chemokines and lymphokines, as recognition and adhesion molecules for cellular and microbial ligands, and as regulators of cell signaling and gene expression. They determine and modulate homeostatic interactions with the host, contribute to innate and adaptive immunity, and mediate many of the pathological consequences of deficient or excessive activation. Some general aspects of macrophage heterogeneity are summarized and the differential expression of macrophage antigens is considered in this chapter as a function of tissue localization and cellular activity. Some of the differentiation antigens expressed by murine monocytes and macrophages are discussed—namely, EGF-TM7 antigens, IgSF antigens, and lectins. There are a number of molecules that function as scavenger receptors, several of which lack collagenous domains. Two such well-characterized antigens that play crucial roles in macrophage biology are scavenger receptor and MARCO.

EGF-TM7: a novel subfamily of seven-transmembrane-region leukocyte cell-surface molecules

Abstract The recent cDNA cloning of three cell-surface molecules has identified a subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seren-transmembrane (Tm7) region. Here, Andrew McKnight and Siamon Gordon review the unusual structural properties of these molecules, their similarities to certain peptidic hormone receptors, and speculate as to their immunological function

Sequence of rat interleukin 2 and anomalous binding of a mouse interleukin 2 cDNA probe to rat MHC class II-associated invariant chain mRNA

We now report the isolation and sequence of a rat IL-2 cDNA clone and the characterization of a cross-reaction to rat class II major histocompatibility complex (MHC)-associated invariant chain mRNA observed when using a mouse IL-2 cDNA probe to analyze rat mRNA

MHC Class II

This chapter provides an overview of tissue distribution, structure, and functions of major histocompatibility complex Class II (MHC Class II) antigen. MHC Class II molecules are expressed on dendritic cells, B cells, monocytes, macrophages, myeloid, and erythroid precursors and some epithelial cells. MHC Class II is expressed on activated T cells in humans and rats. Expression of MHC Class II is regulated by cytokines including interferon γ, which also induces expression on fibroblasts, epithelial, and endothelial cells. MHC Class II molecules are heterodimers of noncovalently associated α and β chains. Both chains are composed of two IgSF domains and have transmembrane sequences and short cytoplasmic tails. MHC Class II molecules present exogenously derived antigen to CD4 + T lymphocyes, which are usually helper T cells. MHC Class II molecules expressed on thymic stromal cells play a key role in the positive and negative selection of CD4 + T cells during thymopoiesis, and genetically engineered mice that do not express MHC Class II antigens lack CD4 + T cells in the periphery. Signaling can occur through MHC Class II.

MHC Class I

This chapter discusses tissue distribution, structure, and functions of major histocompatibility complex Class I (MHC Class I) antigen. The “classical” MHC Class I molecules (HLA-A, -B, and -C in man) are expressed on most nucleated cells, but the expression varies on different cell types. Interferons α, β, and γ and tumor necrosis factor α increase the expression of MHC Class I molecules. The expression can be low on virus-infected or tumor cells. “Nonclassical” MHC molecules generally have a broad distribution. HLA-G is expressed only on cytotrophoblasts. MHC Class I molecules consist of heterodimers of highly polymorphic α chains noncovalently associated with the invariant β 2 -microglobulin subunit. β 2- Microglobulin and the α3 domain are Ig-related and of the C1-set. The “classical” MHC Class I molecules present endogenously synthesized peptides to CD8 + lymphocytes, which are usually cytotoxic T cells. MHC Class I molecules expressed on thymic epithelial cells regulate the positive and negative selection of CD8 + T cells during T cell maturation. Expression of Class I molecules depends on the expression of fig-microglobulin, and mice lacking a functional β2-microglobulin gene do not express Class I molecules on the cell surface. These animals lack mature CD4 − CD8 + T cells and have defective cell-mediated cytotoxicity. Recognition of MHC Class I by NK receptors can protect from lysis by NK cells.

Chromosome mapping of the Emr1 gene

Species: Mouse Locus name: EGF-module-containing mucin-like hormone receptor 1 Locus symbol: Emrl Map position: The distal region of mouse Chromosome (Chr) 17: (centromere) D17Mit7-5.3 + 2.1 cM-Emrl-2.6 +_ 1.5 cM-Hprtpsl. Method of mapping: DNA from two parental strains [C3H/HeJgld and (C3H/HeJ-g/d x Mus spretus)F1] were digested with various restriction endonucleases and hybridized with an F4/80 cDNA probe to determine restriction fragment length variants (RFLVs) to allow haplotype analyses. Database deposit information: MGD-JNUM-40392 Molecular reagents used for mapping: cDNA probe derived from clone pF4/80(12.2) Method of allele detection and nature of aUelic variant: BglII RFLVs were detected and analyzed: C3H/HeJ-gld 11.0 kb, 6.7 kb, and 3.4 kb; Mus spretus 16.0 kb and 8.2 kb. In each of the backcross mice either the C3H/HeJ-gld parental bands or all five bands (two Mus spretus bands and three half-intensity C3H/HeJ-gld bands) were observed, indicating that a single locus was detected. Previously identified homolog: EMR1 (human); Chr 19p13.3 Discussion: F4/80 is a 160-kDa glycoprotein expressed on certain populations of mouse tissue macrophages [1,2]. The isolation of cDNA clones encoding F4/80 demonstrates that the molecule comprises three subregions: a seven transmemhrane-spanning (TM7) region, a membrane-proximal extracellular region abundant in serine and threonine residues, and an NHz-terminal region consisting of seven epidermal growth factor (EGF) domains [3]. This novel structure defines a subset of leukocyte-restricted TM7 molecules designated the EGF-TM7 family, which differ in their number of NHz-terminal EGF domains, the other characterized members of which are human CD97 and human EGF-module-containing mucin-like hormone receptor 1 (EMR1) [4]. The human CD97 gene has been mapped to chromosomal region 19p13.12-13.2 [5], and the EMR1 gene, which is the predicted human homolog of mouse F4/80, maps to region 19t>13.3 [6]. This has led to the prediction for clustered EGF-TM7 genes located on the short arm of human Chr 19. To extend our characterization of the mouse F4/80 gene, we undertook gene mapping studies described in this report. In accordance with the human nomenclature, the gene encoding the F4/80 molecule will hereafter be referred to as Emrl. The position of the EMR1 gene on the short arm of human Chr 19 (19p13.3) extends the homology relationship between this region of the human genome and distal mouse Chr 17.

Protein superfamilies and cell surface molecules

This chapter describes the methods for the identification of superfamilies and shows alignments of some sequences to illustrate the key residues that are often conserved in these superfamilies. The term “superfamily” is useful in discussing sequence similarities in general and also when one wants to distinguish them. The main difficulty in identifying superfamily domains and repeats is in their low level of amino acid sequence identity, but many methods have been developed to analyze the data from the various large-scale sequencing projects. The first step is to use a database searching program such as FASTA or BLAST. In many cases, these programs will pick up some of the members of a superfamily that the new protein sequence matches. However, no search program picks up all superfamily members and it is not uncommon for a relationship to be entirely missed. A second approach is to look by the eye, or apply various computer programs. A third method is to make a consensus sequence for a particular domain type. The PROSITE database contains a large compilation of patterns and sites found in protein sequences. These patterns can then be used to search a novel sequence for the presence of domains or to search the databases.