Analysis of Four Circulating Complexes of Insulin-Like Growth Factor Binding Proteins in Human Blood during Aging.

The primary role of insulin-like growth factor binding proteins (IGFBPs) is to regulate availability of IGFs for interacting with receptors, but IGFBPs perform IGF-independent actions as well. The availability and activity of IGFBPs in the circulation is influenced primarily by their concentration and structural modifications, but possibly also by interaction with major plasma proteins such as transferrin, alpha-2-macroglobulin (α2M), and fibrinogen. Four types of circulating IGFBP complexes were examined in this study by immuno- and ligand-binding assays in adults of different age. The amounts of IGFBP-3/transferrin and IGFBP-1/fibrinogen complexes were similar in middle- and old-aged persons, whereas the amounts of IGFBP-1 (or -2)/α2M monomer complexes were lower in the old-aged group and negatively correlated with total IGFBP-1 (or -2) amounts in blood. In contrast to IGFBP-1, IGFBP-2 was present in significantly greater quantities in complexes with α2M dimer than α2M monomer in older individuals. IGFBP complexes did not bind 125I-labeled IGF-I in amounts detectable by ligand blotting. According to the results of this study, the quantities of IGFBP-1 and IGFBP-2, which interact with α2M, are age-dependent and, in the case of complexes with α2M monomer, they are negatively correlated with the total circulating levels of these two IGFBPs.

[Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

Differential expression of alpha 2 macroglobulin in response to dietylstilbestrol and in ovarian carcinomas in chickens.

BACKGROUND: Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens. METHODS: To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n=5) and DES-treated oviducts (n=5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n=10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses. RESULTS: We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens. CONCLUSIONS: Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens.

Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition.

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.

An alpha2-macroglobulin-serine proteinase complex from human carcinomatous ascites and pleural effusion: isolation, monoclonal antibody preparation, and immunohistochemical study.

A protein with the apparent molecular mass of 720 kDa which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine was purified from ascites and pleural effusion of patients with pulmonary, breast, gastric, and ovarian cancers by chromatographic techniques. When this protein was separated on SDS-PAGE on nonreducing conditions, two bands corresponding to 720 and 360 kDa were seen to have gelatin-digestive activity in zymography assay. Moreover, when it separated by SDS-PAGE on reducing conditions, it migrated as several bands up to 180 kDa. The N-terminal amino acid sequence and immunoreactivity of anti-alpha2-macroglobulin polyclonal antibody revealed that the 180-kDa band was intact alpha2-macroglobulin. The hydrolytic activity of this complex was completely inhibited by diisopropyl fluorophosphate (DFP) and p-amidinophenylmethanesulfonyl fluoride. In addition, the 65-kDa protein observed under reducing conditions bound 3H-labeled DFP. These results suggest that the purified protein is a complex of the plasma proteinase inhibitor alpha2-macroglobulin and a serine proteinase. Several monoclonal antibodies were obtained when the purified complex was used as an antigen. One of these antibodies, which was immunoreactive to this complex but not to alpha2-macroglobulin, gave a positive band corresponding to 65 kDa on SDS-PAGE under reducing conditions. Use of this antibody in immunohistochemical studies revealed immunoreactivities in numerous neoplastic tissues with strong activity in advanced gastric cancers (e.g., poorly differentiated adenocarcinoma). In addition, strong cross-reactivity was detected in glandular cells of the fetus intestine.

Evidence from sequence analysis that hen egg-white ovomacroglobulin (ovostatin) is devoid of an internal beta-Cys-gamma-Glu thiol ester.

53 residues of the internal sequence from the proteinase-binding hen egg-white ovostatin have been determined. The stretch corresponds to residues 945-997 of human alpha 2-macroglobulin. The degree of conservation of residues of the two stretches is approx. 74%. Cys-949, being one constituent of the internal thiol ester site of members of the family of proteins related to alpha 2-macroglobulin, is an Asn-residue in hen egg-white ovostatin, but the other constituent, Gln-952, is preserved. The Cys-to-Asn substitution forms the chemical basis for the lack of thiol esters in hen egg-white ovostatin.

alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region.

alpha 2-Macroglobulin is cleaved by human immunodeficiency virus-1 protease. The cleavage site is the Phe684-Tyr685 bond in the "bait region", an exposed part of alpha 2-macroglobulin, creating the "F-form". The methylamine derivative of alpha 2-macroglobulin is also cleaved at the same bond. The homologous chicken ovomacroglobulin does not form an F-form structure with the protease, although, F-form generation by other enzymes is known. This is possibly due to the lack of a suitable cleavage sequence in the corresponding region of ovomacroglobulin. In human alpha 2-macroglobulin, the interdomain segment between the main part of the molecule and the receptor-binding C-terminal domain is not cleaved by the HIV protease although typical cleavage sequences occur. In AIDS, therefore, HIV protease from infected cells in unlikely to interfere with receptor-binding of alpha 2-macroglobulin.

Physical and chemical characterization of macroglobulin from marine turtle eggwhite.

Macroglobulin was purified from the eggwhite of the marine turtle (Caretta caretta Linn.) by gel filtration through Sephadex G-200 and Sepharose 4B. It was characterized by physical techniques including sedimentation velocity, diffusion and viscosity. Its molecular weight (Mr) was determined as 724,000 with four subunits of equal molecular weight. A large amount of water was hydrodynamically associated with the macroglobulin. It inhibited the activities of trypsin and papain and did not cross-react with human alpha 2-macroglobulin. Its amino acid composition was similar to that of human alpha 2-macroglobulin. Results suggest that turtle eggwhite macroglobulin is a homologous but distinct protein from human alpha 2-macroglobulin.