Steven A. Spencer

Molecular cloning and characterization of human and murine DNase II.

We have cloned and sequenced novel cDNAs that encode human and murine DNase II, the acidic deoxyribonuclease. Sequence analysis predicts that huDNase II contains an N-terminal signal sequence and that mature DNase II has 344 residues with a calculated molecular mass of 38 032 Da. DNase II is a novel enzyme with no homologies to proteins of known function. Surprisingly, C. elegans appears to possess a family of DNase II homologs. Unlike DNase I-like enzymes that have tissue-specific expression patterns, huDNase II is ubiquituously expressed at low levels. When huDNase II is expressed in human 293 cells, we observe secretion of a novel 42-44 kDa glycoprotein; approximately 20-30% of recombinant human DNase II activity is secreted in this system. The secreted enzyme possesses DNA hydrolytic activity and shares biochemical properties with purified DNase II obtained from other species. We also show that the mechanism by which DNase II cuts DNA is similar to DNase I in that the enzyme produces nicks rather than double-strand cuts.

Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages

We have cloned human and murine DNase I-like cDNAs, termed LS-DNase, which are expressed at high levels in liver and spleen tissues. LS-DNase expression is highly specific to macrophage populations within these and other tissues. Mature LS-DNase from both species is a secreted, non-glycosylated protein containing 285 residues, with a calculated molecular mass of 33 kDa and a basic isoelectric point. Human and murine LS-DNase are highly conserved and share 83% identity. Sequence analysis reveals that LS-DNase shares 46% amino acid sequence identity with DNase I. However, several residues identified as important for interaction of human DNase I with actin are not conserved in both human and murine LS-DNase. Consistent with this observation, recombinant human LS-DNase possesses a DNA hydrolytic activity which, unlike DNase I, is not inhibited by G-actin. The existence of a family of DNase I-like molecules that have tissue-specific expression patterns and the possible role of a macrophage specific DNase are discussed.

Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the αl subunit of lymphocyte function-associated antigen-1

The lymphocyte function‐associated antigen‐1 (LFA‐1) binding of a unique class of small‐molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule‐1 (sICAM‐1) and A‐286982, which respectively define direct and allosteric competitive binding sites within LFA‐1s inserted (I) domain. All three molecules antagonized LFA‐1 binding to ICAM‐1‐Immunoglobulin G fusion (ICAM‐1‐Ig) in a competition ELISA, but only compound 3 and sICAM‐1 inhibited the binding of a fluorescein‐labeled analog of compound 3 to LFA‐1. Compound 3 and sICAM‐1 displayed classical direct competitive binding behavior with ICAM‐1. Their antagonism of LFA‐1 was surmountable by both ICAM‐1‐Ig and a fluorescein‐labeled compound 3 analog. The competition of both sICAM‐1 and compound 3 with ICAM‐1‐Ig for LFA‐1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross‐linking studies with a photoactivated analog of compound 3 localized the high‐affinity small‐molecule binding site to the N‐terminal 507 amino acid segment of the α chain of LFA‐1, a region that includes the I domain. In addition, cells transfected with a variant of LFA‐1 lacking this I domain showed no significant binding of a fluorescein‐labeled analog of compound 3 or ICAM‐1‐Ig. These results demonstrate that compound 3 inhibits the LFA‐1/ICAM‐1 binding interaction in a directly competitive manner by binding to a high‐affinity site on LFA‐1. This binding site overlaps with the ICAM‐1 binding site on the α subunit of LFA‐1, which has previously been localized to the I domain.

Growth hormone receptor and binding protein

Publisher Summary The purification and cloning of the growth hormone (GH) receptor and the binding protein have contributed to our understanding of hormonally controlled growth in mammals. This chapter discusses the purification and cloning of growth hormone receptor and binding protein. The GH and prolactin receptors constitute a new family of signaling proteins with a single transmembrane domain. The ability of GH to promote whole-body growth has been demonstrated clinically in the treatment of GH-deficient children and in animals. The lack of easily demonstrated GH effects in cell-based assay systems has led to the somatomedin hypothesis according to which GH produced by the pituitary acts on the liver to induce synthesis and secretion into the circulation of insulin-like growth factor I (IGF-I), which is responsible for skeletal and tissue growth. The availability of the cloned receptors allows elucidation of the intracellular signaling mechanism for these hormones. In situ hybridization work suggests that the GH receptor is widely distributed on many tissues, although it is most abundant in the liver. This wide distribution suggests that direct actions of GH could play a role in the growth process instead of the indirect action suggested by the somatomedin hypothesis. The availability of the GH receptor expressed by recombinant DNA techniques have been useful in determining the amino acids in GH required for binding to the receptor.

Purification and partial sequence of the rabbit mammary gland prolactin receptor.

1. The prolactin receptor from rabbit mammary gland was purified to near homogeneity using a novel hydrophobic interaction chromatographic procedure. 2. Part sequencing (101 residues) revealed 34% identity with the rabbit liver growth hormone receptor, providing support for the existence of a new class of transmembrane receptors regulating growth and lactation.

GROWTH HORMONE RECEPTORS AND BINDING PROTEINS

Growth hormone receptors are found in a wide variety of tissues and are thought to mediate the various actions of growth hormone. Recently, a growth hormone binding protein was demonstrated in serum and shown to have antigenic identity with the liver growth hormone receptor by use of a panel of monoclonal antibodies to the receptor. Here we describe the purification, part sequence and cloning of the rabbit liver growth hormone receptor. Purification and N terminal sequence analysis of the rabbit serum binding protein for GH showed it to be identical to the extracellular region of the rabbit liver GH receptor. Rabbit liver receptor and human binding protein sequences were expressed in COS-7 cells and shown to display predicted hormone specificity and antigenic characteristics. Finally, the rabbit mammary gland prolactin receptor was purified and part sequenced. This showed 34% homology with the rabbit liver GH receptor, and therefore constitutes the second member of a new class of transmembrane receptors regulating growth and lactation.