Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

Ultrastructural studies of human and rabbit alpha-M-globulins.

Electron micrographs of isolated human alpha(2)M-molecules, obtained by the negative contrast technique, revealed morphologically homogenous structures resembling a graceful monogram of the two letters H and I. The modal values for the length and width of the alpha(2)M particles were 170 A and 100 A, respectively. Purified rabbit alphamacroglobulins contained about 80% alpha(1)M- and 20% alpha(2)M-globulins. The isolated rabbit alpha(1)M- and alpha(2)M-molecules were morphologically indistinguishable from one another and from human alpha(2)M-molecules. Preliminary immunoprecipitation studies demonstrated that the two rabbit alphaM-globulins were antigenically different. Sedimentation constant determinations gave s(20, w) values of 18.8 and 18.2 for rabbit alpha(1)M and alpha(2)M, respectively.

Phylogen of immunoglobulin structure and function. 3. Immunoglobulins of the chicken.

Chicken 7.1S immunoglobulin was purified from whole chicken serum by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The macroglobulin was purified by a combination of salt precipitation and Sephadex G-200 gel filtration. Both immunoglobulin molecules yielded 75% heavy (H) chains and 25% light (L) chains when subjected to extensive reduction and alkylation followed by gel filtration in 5 M guanidine-HCl. Antigenically reactive H and L chains were obtained by partial reduction and alkylation followed by gel filtration in 5 M guanidine-HCl. The 7.1S and 16.7S immunoglobulin H chains were antigenically unrelated to each other, whereas the L chains were antigenically indistinguishable from one another. The 16.7S H chains were found to have a mass of approximately 70,000, and the 7.1S H chains had a mass of 67,500. The mass of the L chains was approximately 22,000. Sedimentation equilibrium studies of the 7.1S immunoglobulin molecule gave a mol wt of approximately 170,000 which is in good agreement with the 179,000 predicted on the basis of 2 H and 2 L polypeptide chains. The 16.7S molecule was shown to have a mol wt of approximately 890,000. A reductive subunit that has a mol wt of approximately 174,000 has been isolated from the 16.7S molecule. These values are consistent with the chicken macroglobulin having five subunits, each of which has 2 H and 2 L chains. The hexose contents of the chicken 7.1S and 16.7S immunoglobulins are 2.2% and 2.6%, respectively. The extinction coefficients of the 7.1S and 16.7S immunoglobulins were 13.18 +/- 0.04 and 12.72 +/- 0.77, respectively, when measured in 0.3 M KCl. Based upon physical-chemical and antigenic characteristics, the 16.7S immunoglobulin most closely resembles IgM of mammals. The 7.1S immunoglobulin definitely belongs to a different class than the 16,7S immunoglobulin, but it does not align itself very well with any of the mammalian immunoglobulins. We propose that this molecule be designated as IgY. Furthermore, this designation would be useful for the immunoglobulins of other species for which there is insufficient correlation with any of the known human immunoglobulins.

Idiotypy of rabbit antibodies. II. Comparison of idiotypy of various kinds of antibodies formed in the same rabbits against Salmonella typhi.

The idiotypic patterns detected in the anti-Salmonella typhi serum of one given bleeding have been sought in other bleedings of the same rabbit. Idiotypic patterns carried by antibodies of the second bleeding are not always found in those of the first bleeding. Bifurcated precipitation zones in gels (double diffusion in cells) have been observed in the reaction of several anti-idiotypic sera with the sera of the first and second bleedings of three rabbits, and apparently indicate that idiotypic patterns which were carried by the same molecule in the first bleeding were carried by two separate molecules in the second bleeding. In a comparison of idiotypy of two anti-S. typhi serum samples of one rabbit, the collection of which had been separated by a very long interruption of the immunization, all the idiotypes detected in the early bleeding were also detected in the late bleeding; several idiotypes were detected in the serum of the late bleeding and not in that of the early bleeding. Idiotypy has been observed and compared in IgM and in IgG antibodies. There are, in each of these two classes, antibodies that are not distinguished from antibodies of the other class by anti-idiotypic sera, and consequently that carry the same idiotypic patterns. A comparison has been made between the idiotypy of the two kinds of antibodies which, in anti-S. typhi sera, are precipitated and not precipitated by the polysaccharide. Certain idiotypic patterns are carried only by the antibodies of the first kind. There are also idiotypic patterns which are carried by antibodies of these two kinds, among which certain anti-idiotypic antibodies do not discriminate. Possible cellular implications of observations made on idiotypy at different stages of the immunization of one individual have been discussed.

Monoclonal macroglobulinemia in NZB-NZW F1 mice.

Serum monoclonal macroglobulins were detected in over 30% of NZB/NZW F(1) mice greater than 11 mo of age. The monoclonal nature of the IgM was shown by restricted electrophoretic mobility, characteristic appearance on immunoelectrophoresis, restriction to a single light chain type, and ability to induce anti-idiotypic antisera. The monoclonal macroglobulins were separated from antibodies to DNA and RNA that migrated in the 7S region of sucrose gradients. Enlarged lymph nodes were often present in mice with monoclonal IgM, and a transplantable IgM-producing lymphoid tumor was established from the spleen of one animal.

A human monoclonal macroglobulin with specificity for alpha(2----8)-linked poly-N-acetyl neuraminic acid, the capsular polysaccharide of group B meningococci and Escherichia coli K1, which crossreacts with polynucleotides and with denatured DNA.

We have described an IgM antibody from a patient with macroglobulinemia specifically reacting with poly-alpha(2----8)N-acetyl neuraminic acid (NeuNAc) the capsular polysaccharide of two important human pathogens, group B meningococcus and E. coli K1. This antibody has a narrowly defined specificity in its interactions with polysaccharides, being unable to bind poly-alpha(2----9)NeuNAc or alternating poly-alpha(2----8)alpha(2----9)NeuNAc. However, it shows interesting crossreactivity with seemingly unrelated polynucleotides and denatured DNA, supporting the hypothesis that charged groups with a given spacing may determine the specificity of antigen-antibody interactions on otherwise dissimilar molecular structures. Despite the crossreactivity with denatured DNA and polynucleotides, the antibody does not appear to have adverse effects in the patient. The antibody protects newborn rats against E. coli K1 infection, as well as the standard horse antiserum H46, and one would expect it to prove useful in humans as an adjunct to antibiotic therapy in infections with group B meningococcus and E. coli K1. We have attempted to clone the antibody-producing cells from peripheral blood, and have shown that the relevant cells are present and can be cultured.

Antigenic specificities related to the cold agglutinin activity of gamma M globulins.

Certain antiserums prepared against isolated cold agglutinins demonstrate specificity for gamma M globulins with this activity and not for similar gamma M globulins lacking such activity. The cold agglutinin specific antigens fall into several major and minor groups. Their exact relation to the presumed antibody-combining sites remains to be determined.

Genetic polymorphism of C'3(beta1C-globulin) in human serum.

Genetic polymorphism of the third component of human complement and its breakdown products has been detected in human serum by high-voltage starch-gel electrophoresis. Six phenotypes were observed in a study of 113 randomly chosen Caucasians. Their inheritance is controlled by four codominant alleles at an autosomal locus. The gene frequencies in this study were C3(1), 0.21; C3(2), 0.77; C3(3), approximately 0.01; and C3(4), approximately 0.004.

Macroglobulin structure: homology of mu and gamma heavy chains of human immunoglobulins.

The amino acid sequence of fragments obtained by cyanogen bromide cleavage of the mu-chain of a human gammaM-globulin is homologous to the NH(2)-terminal sequences of the gamma-chain of human and rabbit gammaG-globulins and is related to that of human light chains. This supports the hypothesis that light and heavy chains evolved from a common ancestral gene.

Macroglobulin structure: variable sequence of light and heavy chains.

The variable regions of the light and heavy chains on the same macroglobulin (immunoglobulin M) molecule are no more related in amino acid sequence than are the variable regions of the light and heavy chains of different immunoglobulin molecules. Subgroups of micro chains are similar in their variable sequence to subgroups of gamma chains.

Serum alpha globulin fraction: survival-and-recovery effect in irradiated mice.

An alpha macroglobulin fraction (19S) was isolated from the serum of rats and BC(3)F(1) mice by zonal ultracentrifugation. Both the isologous and heterologous macroglobulin fractions increased survival among BC(3)F(1) mice x-irradiated with 750 roentgens. The mouse macroglobulin fraction also enhanced radiation recovery of hematopoietic tissue as measured by colony-forming assay and iron-59 incorporation into erythropoietic cells. The overall difference in hematopoietic activity in the irradiated (400 roentgens) mice treated with the macroglobulin fraction, in comparison with this activity in the controls, was three- to fivefold in the bone marrow and nine- to tenfold in the spleen between days 4 and 7 after irradiation. This effect was not obtained with the isologous serum protein fraction containing proteins of smaller molecular weight.

Antibiodies of the IgG, IgM, and IgA classes in newborn and adult sera reactive with gram-negative bacteria.

Umbilical cord serum and adult serum antibodies reactive with heat-stable somatic antigens of Gram-negative bacteria (Neisseria gonorrhoeae, Escherichia coli, and Salmonella typhosa) were assayed by using an indirect fluorescent antibody test. Reactive IgG, IgM, and IgA antibodies were identified by using fluoresceinconjugated antisera specific for these immunoglobulin classes.IgG antibody titers in cord serum approximated those found in the corresponding maternal sera. IgM and IgA antibodies were present in adult sera but were not demonstrable or were present only in small amounts in cord sera. The presence of IgG and IgM antibodies reactive with Gram-negative bacteria was confirmed by the testing of purified 7S and 19S fractions. In addition, both IgG and IgM reactivities were inhibited by the prior incubation of serum with purified specific lipopolysaccharide preparations. The ubiquity and magnitude of these natural IgG antibodies in the sera of both adults and neonates have apparently eluded detection in previous studies. The use of bactericidal and agglutination tests, which are apparently more sensitive to the presence of IgM than to IgG antibodies, may account for the failure of previous studies to detect adult and cord IgG antibodies reactive with somatic antigens of Gram-negative bacteria. The presence of these IgG antibodies may be correlated with the resistance to infection demonstrated by most newborns as they are challenged by the septic extrauterine environment.

Immunoglobulins and complement components in synovial fluid of patients with acute rheumatic fever.

Three components of complement and six other serum proteins were assayed in synovial fluid and serum samples from 25 patients with acute rheumatic fever in Trinidad. The resulting data indicate a relative decrease in both early and late components of complement within the synovial fluids which suggests local activation by immune complexes. Such activation of complement within the joint spaces may play a primary role in development of the inflammatory arthritis of acute rheumatic fever.

Is serum protein electrophoresis useful in separating the "high risk group" in patients with colonic polyps?

BACKGROUND: Adenomas have the highest potential or clinical value from among colonic polyps of developing into adenocarcinoma. The aims of this paper are: to establish criteria to identify the high risk group of patients in a group of patients with colonic polyps, to work out a simple scheme for follow-up care after endoscopic polypectomy, and to establish indications for surgery. The usefulness of determination of electrophoresis of serum proteins has been specially analysed to detect early development of malignant growths in patients with colonic polyps regarding alfa-1/alfa-2 and alfa/beta. 67 cases - 21 women, 46 men were tested. Follow-up endoscopy with the electrophoresis was performed after 6 weeks, 6 and 12 months after polypectomy. 97 polyps were resected with endoscopy in 67 patients. 38 patients (39.17%), those constituting the high risk group, were selected. Included were all polyps with grade II and III of cellular differentiation. CONCLUSIONS: 1) alfa-1/alfa-2 and alfa/beta is a helpful test in identifying the high risk group among patients with colonic polyps and it can be used as a screening test, 2) the determination of beta-2-macroglobuline is not useful in the diagnosis of this group of patients, 3) the electrophoresis of proteins should be the first test to perform on patients with colonic polyps. The relation of electrophoresis to endoscopic polypectomy aids evaluations of patients specially predisposed to malignant.

IgM antibody to a tumor-associated antigen (AG-4) induced by herpes simplex virus type 2: its use in location of the antigen in infected cells.

Rate-zonal centrifugation was used to separate 7S and 19S lg's from sera of patients with cervical cancer and of controls. The lg's were assayed for their ability to recognize AG-4, a tumor-associated antigen, and herpes simplex virus 2 (HSV-2) by various serologic assays including neutralization, immunofluorescence, and complement fixation. These studies indicated that antibody to AG-4 is a macroglobulin; antibody-fixing complement with HSV-2 is a 7S lg. The results of various serologic assays did not show absolute correlation; various tests preferentially identified some antigen-antibody reactions. We obtained evidence that AG-4 is the cytoplasm and is probably on the cell surface.

Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy.

The structural change that occurs in alpha-2-macroglobulin upon its interaction with methylamine or chymotrypsin was studied by high-performance gel chromatography and electron microscopy. The result enabled us to estimate the Stokes radius of the protein as 8.8 nm and 7.9 nm before and after binding with the proteinase, respectively. The methylamine-treated protein also had the Stokes radius of 7.9 nm. Similar studies on the chicken and crocodilian ovomacroglobulins showed that these homologues of alpha 2-macroglobulin had Stokes radii of 9.2-9.3 nm and 8.5-8.7 nm before and after binding with chymotrypsin. Their Stokes radii did not change as a result of the methylamine treatment. Electron micrographs of the native and altered forms of the three proteins are presented. This study introduces a simple and quantitative method to study the structural change of alpha 2-macroglobulin and its homologues.

High molecular weight forms of placental lactogen: evidence for lactogen-macroglobulin complexes in rodents and humans.

Gel filtration chromatography of maternal plasma from late pregnant hamsters demonstrated that approximately 90% of hamster placental lactogen-II (PL-II) is present as high mol wt (Mr) forms. A major peak of immunoactive hamster PL-II with a Mr of 600,000 and a smaller peak (Mr, 210,000) were observed. The major high Mr form of hamster PL-II in plasma had a Mr of 360,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). High Mr forms also predominate in placental extracts, but differ from the plasma forms. 125I-Labeled monomeric hamster PL-II (Mr, 22,000) formed a disulfide-bonded complex (Mr, 720,000 by gel filtration and 360,000 by SDS-PAGE) when incubated in vitro with serum of male, nonpregnant female, or pregnant hamsters. The in vitro complex was also formed with unlabeled hamster PL-II. Similar high Mr complexes were formed, but to a lesser extent, when 125I-labeled mouse PL-II and human PL were incubated with the homologous late pregnant serum. High Mr complexes (Mr, 360,000 by SDS-PAGE) were also formed when the three 125I-labeled PLs were incubated with purified human alpha 2-macroglobulin. The most prominent circulating high Mr form of hamster PL-II had a Mr similar to that of alpha 2-macroglobulin by both gel filtration and SDS-PAGE. In addition, it had a mobility similar to that of alpha 2-macroglobulin on nondenaturing polyacrylamide gels, and it was about 10% asparagine-linked carbohydrate by weight, as is alpha 2-macroglobulin. These similarities and the ability of hamster PL-II to form a disulfide-bonded complex with alpha 2-macroglobulin in vitro suggest that the major circulating form of PL-II in the hamster may be a disulfide-bonded complex of one or more PL monomers with alpha 2-macroglobulin or a related plasma protein. Similar complexed forms of PL may be present in mice and humans.

The effeCt of vitamin c supplements on serum cholesterol, coagulation, fibrinolysis and platelet adhesiveness.

The administration of ascorbic acid (1g/day) to healthy adults did not significantly influence the levels of serum cholesterol, plasminogen activator activity, plasminogen, fibrinogen, FR-antigen, partial thromboplastin time, platelet adhesiveness, a-1-antitrypsin or a-2-macroglobulin over the 3-month period of study.

Quantitation of proteins by densitometric scanning of immunoelectrophoretic precipitates.

Densitometric scanning of stained immunoelectrophoretic precipitates of nine human serum proteins showed that the optical density of the precipitates is approximately proportional to the molar concentration of antigen in the sample. Deviations from this correlation were noted for the macroglobulins (mol. wt. greater than 15 x 10(4)). The resolving capacity of the line immunoelectrophoretic procedure permits densitometric valuations of composite patterns.