Circular dichroism studies of M and N blood group-specific glycoproteins and glycopeptides.

CD spectra of M and N blood group glycoproteins and their NH2-terminal tryptic glycopeptides were compared. The CD spectra of glycoproteins obtained suggest the presence of a low content of alpha-helix. The M glycoprotein contained a higher percentage of a periodical structure. CD spectra of M and N tryptic glycopeptides, both sialoglycopeptides and desialylated preparates, were similar and showed positive ellipticities in the range of 210--240 m. However, the spectra of desialylated glycopeptides were red-shifted in respect to spectra of sialoglycopeptides. The CD spectra of glycopeptides suggest the presence of beta-turns.

Immunoglobulins of colostrum--VI. Comparative studies of cytophilic properties of bovine serum and colostral IgG.

In our previous studies, using physical-chemical and serological methods, substantial differences between bovine serum and colostral IgG, especially IgG2, have been shown. The structural differences were localized in the Fc region of immunoglobulins studied. The present comparative studies were undertaken to determine whether structural differences in the Fc region of bovine serum and colostral IgG are reflected in the interaction of these immunoglobulins with the guinea-pig peritoneal macrophage Fc gamma receptor. It was found that binding of bovine serum and colostral IgG1 was a saturable process and only quantitative differences in the mode of binding to the Fc receptor were observed. There is, however, a big difference in cytophilic activity of bovine IgG2--no saturable and reversible binding is observed in the case of bovine serum IgG2.

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization.

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.

Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory properties.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. To study the relationship between the structure of PRP and its immunomodulatory properties, the polypeptide was digested by chymotrypsin. Products of the proteolysis were separated by gel filtration and three fractions were obtained: PRP-1, PRP-2 and PRP-3. The activity of the fractions was compared with the activity of the untreated PRP. It was found that PRP-1 was inactive, whereas PRP-2 and PRP-3 showed an activity in the regulation of the immune response assayed by measurement of PFC, and by studying effects on delayed hypersensitivity, formation of autologous rosette-forming cell, and sensitivity of thymocytes to hydrocortisone. The activity of PRP-2 and PRP-3 was comparable to the activity of PRP. The PRP-3 fraction of low mol. wt was further purified and a pure nonapeptide of mol. wt 1000 (PRP-3b) was isolated. The amino acid sequence of PRP-3b was: Val--Glu--Ser--Tyr--Val--Pro--Leu--Phe--Pro. The nonapeptide showed the full spectrum of biological activities of PRP. Comparison of terminal amino acid suggested that PRP-3b was neither the NH2- nor the COOH-terminal fragment of PRP. The amino acid sequence of the nonapeptide indicated that PRP-3b is different from other known immunomodulators.

Immunoregulatory properties of synthetic peptides, fragments of a proline-rich polypeptide (PRP) from ovine colostrum.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.

Interaction of IgG immunoglobulins with the guinea pig peritoneal macrophage Fc gamma receptors. Effect on the association of the receptors with the membrane skeleton and the cytoskeleton.

Binding of ligands to cell surface receptors may induce an interaction of the receptors with the cytoskeleton and/or membrane skeleton and decrease the solubility of the receptors in nonionic detergents. Cytochalasins, reagents affecting the structure of microfilaments, inhibit some cell functions induced by cross-linking of the receptors with ligands. Information concerning the function of the cytoskeleton in insolubilization of Fc gamma receptors (Fc gamma R) and in Fc gamma R-mediated signal transmission is rather limited. The aim of this work was to investigate the effect of binding of homologous (guinea pig IgG1 and IgG2) and heterologous (rabbit IgG) immunoglobulins to guinea pig peritoneal macrophages on association of the macrophage Fc gamma receptors with the membrane skeleton and cytoskeleton. Cross-linking the macrophage Fc gamma receptors with immunoglobulin ligands induced insolubilization of the receptors in nonionic detergents suggesting association of the receptors with the membrane skeleton and the cytoskeleton. The ligands showed differential effects depending on a subclass and origin of the IgG used. The process of association of the Fc gamma receptors with the skeletons was fast and did not depend on temperature. Treatment of insoluble complexes with cytochalasin D, DNAse I or colchicine showed that actin microfilaments and microtubules play a role, at least partially, in insolubilization of the cross-linked macrophage Fc gamma receptors. Inhibition of insolubilization of the macrophage Fc gamma receptors by genistein indicated that tyrosine kinases are involved in the process of insolubilization. The association with the skeletons might be a part of the process of transduction of a signal which depended on the subclass and origin of IgG used and on the type of the Fc gamma receptor.

Cell surface sialic acid affects immunoglobulin binding to macrophages.

We demonstrate that guinea pig peritoneal macrophages pretreated with neuraminidase from Vibrio cholerae bind more 125I-IgG than non-treated cells. Estimation of binding constants (Ka and Bmax) shows that the elevation of binding is the result of an increase in affinity and not in the number of receptors for IgG. The change of affinity is proportional to amounts of sialic acid liberated from the cells by increasing doses of neuraminidase. It is also shown that affinity of interactions of IgG with the macrophage receptor is pH dependent. These results indicate that electrostatic forces are important for IgG binding to the macrophage Fc gamma R. The IgG-Fc gamma R interaction can be modulated by changing the degree of sialylation of the macrophage surface glycoconjugates.

A mild method for the purification of guinea pig peritoneal macrophage Fc gamma receptors. Affinity chromatography and elution of the receptor with reducing agents.

A method for the purification of Fc gamma receptors from guinea pig peritoneal macrophages using mild conditions is described. The method is based on the observation that reduction and alkylation of IgG disulfide bonds partially or completely abrogate their binding to Fc gamma receptors. Cell lysates were directly applied to Sepharose-IgG or Sepharose-TNP-Ab(IgG) and the specifically bound Fc gamma receptor was eluted from adsorbents by incubation with reducing agents (2-mercaptoethanol or dithiothreitol). Alternatively, cell lysates were first treated with IgG and then applied to Protein A-Sepharose and the receptor was eluted with reducing agents. Yields of the purified Fc gamma receptor preparations and their activities were considerably higher than when the receptor was eluted from affinity chromatography gels with acetic acid or other acidic buffers or chaotropic agents. The best results were obtained when Fc gamma receptor-IgG complexes were applied to Protein A-Sepharose. No significant difference in the subunit structure was observed using SDS-PAGE when receptor preparations obtained by elution with reducing agents were compared with preparations obtained by elution with acidic buffers.

Human placental membrane receptor for IgG. Purification of the receptor and its subunit structure.

The method of purification of the human placental Fc receptor to an active form is described. The FcR was purified from the glycoprotein fraction of the placental membranes by immunoprecipitation and chromatography on DEAE-cellulose. The purified FcR corresponded to 1.5-2% of the protein present in the crude glycoprotein fraction (PGP) and showed the tendency to aggregate. In the presence of 1% SDS, 4 M urea or 5 M guanidine- HCl the placental FcR dissociated into subunits of molecular weight of 60,000-65,000. The 60,000-65,000 dalton glycoprotein subunits regarded as monomers of FcR are composed of two chains of molecular weight 25,000-30,000, linked by disulphide bonds. The subunits, after removal of dissociating agents, displayed IgG binding activity.

Towards an understanding of biological role of colostrinin peptides.

The aim of this study was to elucidate the structure and possible function of colostrinin, also known as a proline rich polypeptide (PRP). The molecular weight of colostrinin was originally determined by gel filtration to be 17,200 daltons. In the presence of guanidinum chloride, however, the molecular weight was found to be about 6,000 daltons. Further studies utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy revealed that colostrinin is a complex consisting of many low molecular-weight polypeptides. A total of 32 peptides were isolated from the original colostrinin preparation by HPLC and subjected to the N-terminal sequence analysis. The results of sequence analysis revealed significant homology of the peptides to three protein precursors: annexin, beta-casein, and a hypothetical beta-casein homolog. In addition, the sequence of several peptides showed no significant homology to any specific protein in the current GenBank database. The synthetic peptides of various lengths representing the N-terminal sequence of the colostrinin peptides were made to study some biological effects. Here we report that colostrinin and some of its component peptides are potent inducers of leukocyte proliferation and of certain cytokines. Also, a series of monospecific antibodies were produced in rabbits against the synthetic peptides. The antibodies were used to study the kinetic of antigen reduction in colostrum and mature milk following lambing. A threefold decrease was common for most antigens studied over the period of 72 h. Based on the results of these studies we postulate that colostrinin represents a diverse group of peptides produced in the mammary gland of mammals for the development of the optimal physiologic responses in offspring. Also, it is hoped that the beneficial use of colostrinin in Alzheimer's Disease (AD), recently reported elsewhere, will revive interest in its clinical application for treatment and/or prophylaxis of many age-related disorders.

Cognitive effects of Colostral-Val nonapeptide in aged rats.

Colostrinin, a complex of polypeptides derived from sheep colostrum retards the progress of Alzheimer's disease and facilitates acquisition and retrieval of spatial memory in aged rats. Here we investigated the cognitive effects of colostrinin-derived nonapeptide (Colostral-Val nonapeptide, CVNP) in aged rats that demonstrated learning deficits. Administered for 14 days, CVNP did not affect the acquisition of spatial learning or memory retrieval in the Morris water maze. As a result of reversal learning, placebo treated rats shifted searching behavior and swam less in the area of original platform position and more in the area of recent platform position, suggesting formation of the new spatial map. CVNP treated rats did not change the searching pattern and still investigated the area that contained "original" escape platform, suggesting that CVNP treatment delays the extinction of spatial memory. In another experiment, CVNP administered for 8 days did not influence the acquisition of the active avoidance task, but significantly delayed its extinction. The present findings indicate that colostrinin-derived nonapeptide may delay the extinction of long-term memories.

Effect of glycosylation inhibitors on binding properties of the Fc gamma receptors from guinea pig peritoneal macrophages.

Guinea pig peritoneal macrophages possess on their surfaces receptors for the Fc region of IgG immunoglobulins (Fc gamma R). The cells treated with the glycosylation inhibitors tunicamycin or monensin interacted with IgG with a higher affinity and showed a lower number of IgG-binding sites in comparison with control cells. This indicates that the interaction of IgG with the macrophage Fc gamma receptor depends on the degree of glycosylation of the cell surface glycoconjugates and that glycosylation of the macrophage Fc gamma receptor is important for the expression of the mature form of the receptor.

Cytokine-inducing activity of a proline-rich polypeptide complex (PRP) from ovine colostrum and its active nonapeptide fragment analogs.

A complex of proline-rich polypeptides (PRP) was isolated from ovine colostrum in our laboratory and was shown to possess immunomodulatory properties and psychotropic activity, including beneficial effects in the treatment of Alzheimer's disease. A nonapeptide fragment (NP): Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro, isolated from the chymotryptic digestion products of PRP, and its C-terminal fragment, a hexapeptide (HP): Tyr-Val-Pro-Leu-Phe-Pro also exhibited immunoregulatory activity. Although NP and HP expressed activity similar to that of PRP in studies on humoral and cellular immune responses, in other immune processes, e.g. induction of cytokines, they showed markedly lower activity than PRP. In the search for more active peptides, in the present study, we compared the cytokine-inducing ability of PRP, NP, HP, and linear oligomers of NP or HP. For this purpose, the induction of IFN, TNF-alpha, IL-6, and IL-10 in human whole blood cell cultures was measured. NP, HP, and their oligomers showed differential effects in the induction of cytokines, generally lower than that of PRP. Only the PRP complex showed a bell-shaped dose-response dependence suggesting regulatory properties. There were no distinct differences between monomeric forms of NP (NP1) or HP (HP1) and their oligomers in the induction of IFN and TNF-alpha (Th1 cytokines) but such differences were found in the induction of IL-6 and IL-10 (Th2 cytokines). Dimer (NP2) was less active than the monomeric NP1 nonapeptide in the induction of IL-6 and IL-10. On the other hand, oligomers: HP3 and HP4, showed a significantly higher ability to induce Th2 cytokines compared to HP1, HP2 or NP peptides. This was especially evident in the case of IL-10 induction, where the activity of HP4 surpassed the activity of PRP and approached the activity of LPS-PHA. The results obtained showed that some of the peptides studied, when used at higher concentrations (100 microg/ml) may replace the PRP complex as cytokine inducers. Our data also suggest the possibility of using certain oligomers for selective induction of particular cytokines.

Chemical properties of the placental FC gamma receptor.

The carbohydrate and amino acid compositions of the purified Fc receptor from human placental trophoblasts are described. The placental receptor is a glycoprotein. The total content of carbohydrates in the receptor is 30% (w/w). The major carbohydrate components are galactose, N-acetylglucosamine, fucose, and mannose. The placental receptor has a high content of acidic amino acids and a relatively low number of aromatic and hydrophobic amino acid residues. The ratio of contents of hydrophilic to hydrophobic amino acids is 2:1. The hydrophilic nature of the receptor molecules is shown by charge-shift electrophoresis.

Immunoglobulins of colostrum. V. Comparative studies of ovine and bovine serum and colostral IgM.

Comparative studies of physiocochemical properties of ovine and bovine serum and colostral IgM are described. Serologic properties, molecular weight, amino acid composition, carbohydrate content, peptide maps, heterogeneity, and ORD and CD spectra were compared. Colostral IgM have additional antigenic determinants, higher carbohydrate content, lower heterogeneity, and higher content of beta-structure. Different amino acid composition and different peptide maps of serum and colostral IgM were shown. The results obtained suggest that ovine and bovine colostral IgM immunoglobulins may be a mixed population of molecules locally synthesized and transferred from the serum.

Polyclonal antibodies directed against human placental Fcgamma receptor. Characterization of the antibodies and their interaction with the receptor.

Antibodies to the putative Fc gamma receptor (Fc gamma R) of human placenta were raised by immunization of rabbits with the receptor purified form syncytiotrophoblast plasma membranes of human placenta. The rabbit antibodies were of IgG class and their F(ab')2 fragment interacted with Fc receptors in solubilized form and membrane-bound, as well. Immunological reactivity of the antibodies with Fc gamma R was demonstrated using immunodiffusion, solid-phase immunoassay, and ELISA. Studies on interaction of the antibodies with the isolated placental Fc gamma R showed that antigenic determinants of the receptor were different from the IgG-binding site. Rabbit anti-human placental Fc gamma R crossreacted, to various extent, with Fc gamma R-positive human cell lines showing antigenic relatedness of the placental receptor with Fc gamma R on other cell types. The antibodies showed only a weak crossreactivity with guinea pig peritoneal macrophage Fc gamma R. SDS-PAGE analysis of immunoprecipitates obtained by treatment of detergent lysates of 3H-labeled human placental trophoblasts membranes with the rabbit antibodies or with human IgG showed the presence of the some components which were observed in the case of the isolated, purified placental Fc gamma R: Mr of 123,000 and 52,000-56,000 under nonreducing conditions, and Mr of 64,000-67,000, 52,000-56,000, and 26,000-29,000, under reducing conditions. The polypeptide chains of the purified human placental receptor resolved in SDS-PAGE and transferred on nitrocellulose strips were able to interact both with the rabbit anti-placental receptor IgG F(ab')2 fragments and with human IgG. This gives an evidence that human placental Fc gamma R polypeptide chains Mr of approx. 64,000, 54,000, and 28,000 contain antigenic determinants of the receptor and binding sites for the Fc region of IgG, as well.

Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Physicochemical and serological studies on immunoglobulins, their Fab and Fc fragments, L and H chains, showed that the strongest differences occurred in case of IgG2, and that the differences were localized in the Fc region.

Immunoglobulins of colostrum. V. Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Studies on localization of structural differences between ovine and bovine serum and colostral IgG immunoglobulins are described. Comparison of heterogeneity, susceptibility to proteolytic enzymes, peptide maps, amino acid compositions, and antigenic properties of immunoglobulins and their Fab and Fc fragments and H and L chains showed that structural differences are localized in the Fc region. The strongest differences were found in case of IgG2. It was also shown that no Fc fragments could be obtained from bovine serm IgG2 and ovine serum and colostral IgG2 due to their susceptibility to papain and trypsin. The results obtained confirmed our suggestion that colostral IgG2 are locally synthesized in mammary glands, whereas colostral IgG1 might be a mixed population of molecules locally synthesized and transferred from the serum.

Proline-rich polypeptide (PRP)--an immunomodulatory peptide from ovine colostrum.

The structure and properties of a new immunomodulatory peptide isolated from ovine colostrum are described. PRP acts both in vivo and in vitro, and is not species specific. PRP increases permeability of skin vessels, and causes differentiation of murine thymocytes into functionally active T cells. It can simultaneously change surface markers and function of cells. The polypeptide is able to reduce binding of peanut agglutinin (PNA) to PNA+ thymocytes and to increase the binding of PNA to PNA- cells. PRP is also able to transform cortisone-resistant thymocytes into cortisone-sensitive, and vice versa. The observed changes occurred concomitantly, i.e. changes in binding of PNA were accompanied by changes in resistance to cortisone and in expression of helper or suppressor activity. The fact that changes induced by PRP are reversible after the second exposure of the cells to the polypeptide makes it unique among known immunomodulators. An active nonapeptide fragment: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the products of PRP digestion. It shows full spectrum of biological activities of PRP. The sequence-Pro-Leu-Phe- is responsible for the immunological effect of the peptide.

Human placental Fc gamma receptor. Studies on affinity labeling of the receptor with IgG.

The aim of the presented work was to find out whether the Fc gamma receptor from human placental syncytiotrophoblast plasma membranes in its native membrane-bound state is composed of more subunit chains than previously found in our laboratory in the purified receptor. The chains might be lost during the purification procedure with the use of various chaotropic reagents, e.g. acidic or alkaline buffers, detergents. To study this problem, affinity labeling technique and bifunctional crosslinking reagents were used to covalently link IgG with the Fc gamma receptor. The reagents used were: noncleavable dimethylsuberimidate (DMS), cleavable dimethyl 3,3-dithiobispropionimidate (DTBP), and photoactivable sulfosuccinimidyl 6-/4' azido-2'nitrophenylamino/hexanoate (sulfo-SANPAH). Human 125I-IgG were crosslinked to the membrane-bound receptor or unlabeled IgG was crosslinked to 3H-labeled placental membranes. When 125I-IgG were used in the presence of an excess of unlabeled IgG, recovery of radioactivity after crosslinking was much lower, indicating that human IgG and the placental Fc gamma receptor were involved in the formation of crosslinking of ligand-receptor complexes. The products of crosslinking were analyzed in SDS-PAGE. In the absence of reducing agents, high molecular weight products were present which did not enter the gel or formed broad diffused bands at the top of the gel. Therefore, the products of crosslinking were analyzed under reducing conditions. Analysis by SDS-PAGE demonstrated a major polypeptide band Mr of 160,000 in soluble products of crosslinking of IgG to the placental Fc gamma receptor, regardless of which noncleavable crosslinker was used. The protein is built either of two molecules of the receptor subunit chain, Mr of 60,000 and one IgG heavy chain (Mr of 56,000) or of two IgG heavy chains and one molecule of the receptor chain. The presence of this protein in control samples was not observed at all or its content was markedly lower. The same effect was also observed when DTBP, a cleavable crosslinking reagent was used. In this case, 125I-IgG heavy and light chains or the receptor subunit chains were found after SDS-PAGE under reducing conditions. The results presented in this paper do not suggest a presence of additional chains in the placental Fc gamma receptor others than described in our previous paper concerning the subunit structure of this receptor.