Identification and characterization of some serum proteins of the laboratory rat (Rattus norvegicus) by crossed immunoelectrophoresis.

Rat (Rattus norvegicus) serum proteins were examined by means of crossed immunoelectrophoresis. We have performed the topographic characterization of prealbumins, albumin, orosomucoid, glycoprotein of Darcy, alpha-macroglobulins, haptoglobin, hemopexin, transferrin and IgG. There are several differences in rat serum in comparison to human serum. This applies, for example, to prealbunins and to Darcy's glycoprotein. The behaviour of these fractions in different conditions is discussed in more detail.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Circular dichroic spectroscopy of non-human alpha-macroglobulins

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.     

Human fibroblast collagenase-alpha-macroglobulin interactions. Localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins

The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin, and rat alpha 1-inhibitor 3) differing in primary and quaternary structure has been investigated. Complex formation with each of these alpha-macroglobulins follows the course identified for many other proteinases, i.e. specific limited proteolysis in their bait regions inducing a set of conformational changes resulting in activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters and covalent complex formation. At collagenase: alpha-macroglobulin molar ratios of less than 1:1 3.2-3.6 mol of SH groups appear for 1 mol of collagenase bound to human and rat alpha 2-macroglobulin and to rat alpha 1-macroglobulin. For these alpha-macroglobulins it can be estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1 s-1 while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3. More than 95% of the complexed collagenase is covalently bound, and sodium dodecyl sulfate gel electrophoresis shows the typical pattern of bands corresponding to reaction products of very high apparent molecular weight. The same pattern is also seen in the covalent (greater than 98%) complex very slowly formed from Clostridium histolyticum collagenase and human alpha 2-macroglobulin. The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens. These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin, as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues. In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase. It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.     

Carbohydrate recognition systems in amphibians: primitive alpha 2-macroglobulin receptors

Two alpha-macroglobulins were isolated from the plasma of the frog. Murine clearance studies were performed with these proteins after they were reacted with proteinase. These studies indicated that clearance behavior was more complex than observed with avian or human homologues, since it involved not only specific receptors for the complex, but also carbohydrate recognition. Recognition of one of these macroglobulins by both the alpha-macroglobulin receptor and carbohydrate receptors required conformational change in the inhibitor. Clearance studies were then performed in frogs to probe the nature of carbohydrate receptor recognition. Model glycoproteins were employed to avoid the problem of heterogeneous carbohydrate end groups in the alpha-macroglobulins. These studies demonstrated that the N-acetylglucosamine/mannose receptor is the major carbohydrate recognition system in frogs.     

A conserved region in alpha-macroglobulins participates in binding to the mammalian alpha-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The properties of rabbit alpha1-macroglobulin upon activation are distinct from those of rabbit and human alpha2-macroglobulins

We have characterized native and activated forms of rabbit alpha1M and compared them to rabbit and human alpha2M. Similar to human alpha2M, rabbit alpha1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human alpha2M, rabbit alpha1M is cleaved by trypsin at one site; however, rabbit alpha1M shares characteristics with rabbit alpha2M that are different from the properties of human alpha2M. Amine or trypsin treatment of rabbit alpha-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit alpha1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit alpha-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit alpha1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit alpha1M bind to macrophages with similar affinity to that of human alpha2M, trypsin-treated rabbit alpha1M exhibits dramatically lower affinity. This suggests that rabbit alpha1M may not play the same proteinase-inhibiting physiological role as human alpha2M.     

Trasylol prevents trypsin-induced shock in dogs.

The effect of simultaneous intravenous administration in the dog of bovine trypsin and Trasylol followed by continued infusion of Trasylol was studied. Special attention was paid to the interchange between the dominating plasma protease inhibitors alpha1-antitrypsin and a-macroglobulins and to the disappearance of Trasylol and its trypsin complexes from the circulation. The following results were obtained: 1) Trypsin was preferentially bound by the alpha-macroglobulins, though Trasylol is a strong trypsin inhibitor. 2) On saturation of the alpha-macroglobulins, a considerable amount of trypsin was bound by alpha1-antitrypsin. 3) Trasylol was bound to the trypsin-alpha-macroglobulin complexes and then rapidly eliminated from the circulation. 4) On saturation of the alpha-macroglobulins, Trasylol was identified in a free form but increasing amounts of Trasylol were also bound to trypsin. This could be explained not only by direct complexation of Trasylol and trypsin but also by a transfer of trypsin from unstable trypsin-alpha1-antitrypsin complexes to free Trasylol.     

Effects of alpha-macroglobulins and murinoglobulins on the hemagglutination by influenza C virus

Rat alpha-1- and alpha-2-macroglobulins as well as rat murinoglobulins I and II were shown to inhibit hemagglutination by influenza C virus. In marked contrast, neither alpha-macroglobulins nor murinoglobulins from mouse or guinea pig plasma had the inhibitory activity. These results suggest that the hemagglutination-inhibiting activity of rat alpha-macroglobulins or murinoglobulins is not related to their protease-binding capacity.     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.     

Homology between the Lpm system of allotypes in the American mink and the Gp system of allotypes in the domestic pig]

The 5 alpha-macroglobulin allotypes alpha M1, alpha M2, alpha M3, alpha M4 and alpha M5 were identified in pig. The alpha M1 allotype was reported as a marker of pig alpha-macroglobulin, the latter being homologous to alpha 2-macroglobulins in human and in mink. The allotypes alpha M2-alpha M5 were specified as markers of the second isotypical variant of pig alpha-macroglobulins, which was homologous to mink Lpm macroglobulin (alpha 1M). As seen from data obtained by International Comparative Test ISABR 87-88, alpha M1 is a new allotype, while allotypes alpha M2--alpha M5 correspond to four allotypes in the Gp system (Janik et al.). Based on these data, a conclusion was made on the homology between the Lpm system in american mink and the Gp system in pig. Since the allotypes studied are the part of alpha-macroglobulins, a locus controlling them was designated the AM locus. We also find it more advantageous to apply the same name to the homologous locus in mink, instead of the Lpm used earlier. Genetic control of 5 allotypes was studied and the structure of the AM locus in pig analysed in detail. Comparative study of organization of the above locus and the homologous locus in mink was carried out.     

Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.     

An alpha(2)-globulin component present in sweat, saliva, tears, human milk, colostrum and cerumen

By double-diffusion and immunoelectrophoresis, using rabbit anti-sweat protein immune serum, an alpha(2)-globulin component of glycoprotein character was found in sweat, saliva, tears, human milk, colostrum and cerumen.     

Distribution of alpha 2M-globulin and Lpm-protein in the organs and tissues of the American mink

Distribution in the organs and tissues of two proteins of alpha-macroglobulin fraction, that differ in their antigenic structures, has been studied in the American mink. The both proteins (alpha 2M and Lpm) are present in hepatocytes, in cells of the follicular epithelium of the ovary, in the thymic bodies, in the alveolar macrophages of the lungs and in the splenic lymphoid nodes. Joint localization of alpha 2M and Lpm is revealed in the connective tissue of all the organs examined. The exception make the stomach and the uterine, where alpha 2M is revealed but not Lpm. The results obtained demonstrate a similar distribution of the two alpha-macroglobulins in the mink organism. They correspond to the literature data on morphofunctional topography of alpha 2M in the man. Certain individual differences in alpha 2M and Lpm localization can reflect peculiarities inherent in each of these alpha-macroglobulins of the American mink.     

Serological identification of spectrin in the 2 dimensional immunoelectrophoresis]

Using polyvalent antisera directed against solubilized human erythrocyte ghosts in the immunoelectrophoresis we obtained some precipitation arcs. Antisera against the purified spectrin allowed the identification of spectrin in the crossed immunoelectrophoresis.     

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.     

Alpha-macroglobulin-like plasma inactivator for Vibrio vulnificus metalloprotease.

The metalloprotease produced by Vibrio vulnificus (VVP) is known to be quickly inactivated by plasma proteins which belong to the class of alpha-macroglobulins in vitro at a molar ratio of 1:1. But the in vivo potential of the inactivators has not been studied. Macroalbumin (MA), a member of alpha-macroglobulins in guinea pig plasma, was found to inactivate VVP by means of physical entrapment in vitro. In vivo actions of VVP, permeability-enhancing and hemorrhagic actions, were greatly augmented by simultaneous injection of the antibody against MA, which had no effect on in vitro proteolytic action toward azocasein. The interstitial-tissue space in the normal guinea pig skin contains a negligible amount of MA. However, sufficient MA was present in the extravascular fluid collected after the intradermal injection of VVP. Besides, in the extravascular fluid, VVP formed a complex with MA and no inactivator other than MA was found. These results indicate that plasma MA leaked from the vascular system owing to the permeability-enhancing and hemorrhagic actions of VVP, resulting in inactivation of VVP in situ.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.     

Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha2-macroglobulin.

To better understand the structural basis for the binding of proteinase-transformed human alpha2-macroglobulin (alpha2M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15-N and 15N-enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter- and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein. The beta-strands form three regions of antiparallel beta-sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378). Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.     

A precipitin for human serum proteins as released into the environment by stressed bait shrimp, Penaeus duorarum

1. A precipitin for human serum proteins is released into the environment by stressed bait shrimp, Penaeus duorarum. 2. Two-dimensional crossed immunoelectrophoresis revealed that the precipitin reacts (a) primarily with proteins belonging to the major group, alpha-1 globulin; and (b) with more proteins than a standard mammalian antiserum. 3. This study extends the variety of species known to liberate precipitins and suggests this response may be widespread among invertebrates. 4. The precipitins are easily collected in saline solution and, by virtue of their unique specificities, are potentially useful in diagnostic testing.