No abstract

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-b-1a~IFN-b-1a! were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-b-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-b-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-b-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate~DSG! indicated that the major cross-linked species had an apparent M r consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-b-1a0IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-b-1a0IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent M r consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-b-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-b-1a0 IFNAR2 and IFN-b-1a0IFNAR10IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-b-1a and its receptor. Weber, 1986;Diaz et al., 1994;Pestka, 1997aPestka, , 1997b IFNs mediate their biological activity by binding to the extracellular portions of specific IFN receptors. All of the type I IFNs bind to, and signal through, a common receptor composed of two distinct chains, IFNAR1 and IFNAR2~Uze et al., 1995!, whereas IFN-g binds to, and signals through, its own unique receptor composed of a-and b-chains~Soh et al., 1994; Walter et al., 1995!. Mature IFNAR1~after removal of the peptide leader sequence! is a 530 amino acid residue integral membrane protein~Uze et al., 1990!, while mature IFNAR2 has been isolated as three forms: the full length receptor chain comprised of 487 amino acids~referred to as IFNAR2.2! with 251 residues in the cytoplasmic portioñ Domanski et al., 1995; Lutfalla et al., 1995! and two shorter forms, one comprised of 303 amino acids~referred to as IFNAR2.1! with only 67 residues in the cytoplasmic portion~Novick et al., 1994;Domanski et al., 1995;Lutfalla et al., 1995;Pfeffer et al., 1997! and Abbreviations: CV, column volumes; DSG, dissucinimidyl glutarate; ESI-MS, electrospray ionization-mass spectrometry; IFN, interferon; IFN-b-1a, interferon-b-1a; IFNAR1, human type I interferon receptor chain 1; IFNAR2, human type I interferon re...

An abundant protein that is identical to the growth-associated protein pleiotrophin (PTN) has been isolated from dissociative extracts of bovine nasal and fetal epiphyseal cartilage. The yield from these tissues was at least 15 micrograms/g wet weight of cartilage. PTN was absent or was present only in trace amounts in mature articular cartilage. An analysis of tryptic fragments of PTN, held together with disulfide bonds, did not indicate any set pattern of cystine cross-links, which suggests a propensity for rapid refolding of the protein. PTN could not be isolated from thin (10 microns) slices of nasal cartilage in physiological extraction buffers, which indicates that it was tightly associated with the cell surface, was tightly associated with nonextractable matrix, or was an intracellular protein. Its appearance in various extraction media parallels that of histone H2b, a nucleosomal protein; this suggests a possible intracellular location for the protein. Immunohistochemical analysis of its distribution in fetal epiphysis indicated that it is associated with chondrocytes.

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.