Binding of immunoglobulins to the major progesterone-induced proteins secreted by the sheep uterus

We examined the binding of immunoglobulins to the uterine milk proteins, the major progesterone-induced proteins secreted by uterine endometrium of pregnant ewes. Binding was ascertained by measuring binding of 125 I-immunoglobulin to uterine milk proteins that were Western or dot-blotted to nitrocellulose or were coupled to Sepharose. The magnitude of binding was greatest for sheep IgM, intermediate for sheep secretory IgA, low for human secretory and serum IgA, and barely detectable for sheep IgG. Binding of IgA and IgM to uterine milk proteins was time and concentration dependent, saturable, inhibited by high ionic strength buffers, and lost due to enzymatic destruction of the Fc portion of the immunoglobulin molecule. In conclusion, the uterine milk proteins preferentially bind IgA and IgM in a species-dependent manner. Such binding may be related to the role of these proteins in the uterus and may make the uterine milk proteins a useful tool for studying or purifying sheep immunoglobulins.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

Interaction of oligonucleotides and ATP with preparations of sIgA possessing protein kinase activity

Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.     

A new protein with a particular thermoprecipitability in bovine milk.

A new protein with a particular thermoprecipitability was isolated from bovine milk and tentatively termed milk pyroglobulin. The protein which was soluble at a relatively cold temperature was precipitated by raising the temperature to a certain degree depending on the concentration of the protein. The precipitate disappeared on recooling. This protein had the electrophoretic mobility of gamma globulin but did not carry either antigenic specificities of immunoglobulins or of free secretory component. The molecular weight was estimated to be approximately 60,000 in thin-layer gel filtration on Sephadex G-200 superfine gel, but the protein appeared to be convertible to molecules with a lower molecular weight of approximately 20,000 in the presence of bovine serum albumin. The presence of the albumin inhibited the thermoprecipitation as did alpha-lactalbumin but not IgG immunoglobulin from bovine colostrum. In SDS-polyacrylamide gel electrophoresis, the protein was separated into two components having a molecular weight of 19,000 and 10,000, respectively. Both components were thermoprecipitable and carried identical antigenic determinants.     

Isotype-specific rabbit antibodies against chinchilla immunoglobulins G, M, and A.

Chinchillas have become a preferred animal model for studying otitis media, and are also useful in studying insulin release, gastrin physiology, intestinal infection, and hepatocellular pathophysiology. Immunopathologic studies in the model, however, have been limited by absence of specific antibody reagents against chinchilla immunoglobulins. We describe a method for preparing isotype-specific rabbit antibodies against the heavy-chain components of chinchilla immunoglobulins G, M, and A. Chromatographic techniques were used to isolate chinchilla immunoglobulins from serum and breast milk; heavy-chain fractions were isolated and used as antigens to produce isotype-specific antibodies in New Zealand White rabbits. Enzyme-linked immunosorbent assay of these antisera disclosed anti-light chain cross-reactivity, which was removed by affinity chromatography. The isolation and affinity purification techniques were highly reproducible. The availability of these reagents should greatly enhance the utility of the chinchilla in modeling human disease.     

Raman spectra and conformational structures of Fab mu and (Fc)5 mu fragments of cryoglobulin IgM-kappa McE.

Raman spectra have been obtained on aqueous solutions of the following human immunoglobulins: IgM-kappa McE, IgG-kappa Ger, IgM-kappa WSm and IgG-lambda Gui. The former two species exhibit the property of cryoprecipitation. Comparison of the spectra shows that all immunoglobulins have similar secondary structures, predominantly of the beta-sheet type. Fab mu and (Fc)5 mu fragments of IgM-kappa McE also yield Raman spectra which indicate closely similar secondary structures. Minor differences among the spectra can be explained by differences in amino acid compositions of the respective proteins.     

Temporal changes in milk proteomes reveal developing milk functions

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.     

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.     

Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus.

Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.     

Acceleration of Dextransucrase Activity of Streptococcus mutans by Secretory Immunoglobulin A

The effect of immunoglobulins on the activity of dextransucrase purified from Streptococcus mutans strain HS-6 is described. When human salivary immunoglobulin A (IgA) or colostral IgA, either natured or denatured, was incubated with dextransucrase, the rate of the dextran synthesis was markedly accelerated, whereas human serum IgA or IgG neither accelerated nor inhibited the enzyme activity. The results suggest that a portion unique for secretory IgA, the secretory component, might be related to the enzyme acceleration. On the other hand, specific rabbit antiserum against the dextransucrase inhibited completely dextran synthesis by the enzyme.     

Orientation and kinetics of surface denaturation of normal human and myeloma IgA in monolayers at the interface of air-NaCl aqueous solutions

Normal serum immunoglobulin A (IgA) and abnormal IgA isolated from serum of patients with multiple A-myeloma have been studied by monolayer technique at air--NaCl solution interfaces. Normal IgA analogous to human normal IgG and secretory IgA was shown to have horizontal orientation at air--water interface. Only some abnormal IgA were similar to myeloma IgG and differed from the normal ones by their orientation at phase border. Majority of myeloma IgA under study could not be distinguished from the normal ones by orientation and denaturation kinetics at interface. B-lymphocytes of the first group of patients were assumed to carry IgA-receptors at their surface, but B-lymphocytes of the second group of patients carried Ig receptors of some other class of immunoglobulins.     

Structural analysis of three sulfated oligosaccharides isolated from human milk

The structures of two sulfated octasaccharides and one sulfated nonasaccharide isolated from human milk have been investigated. Using 13C and 1H NMR spectroscopy and ESMS, the following structures 1-3 were established: [formula: see text].     

Number of ways of joining SH groups to form multi-peptide chain proteins

The expression for the total number of isomeric structures formed upon oxidation of SH groups has previously been correctly obtained for single chain proteins only. Expressions have now been obtained for molecules consisting of 2 and 4 peptide chains of the types A2 and A2B2. The latter type is particularly important as exemplified by the immunoglobulins and the insulin receptor. For the oxidation of insulin A chain with 4 SH groups, the total number of isomeric A2 structures is 59--this is different to all the values previously reported. Lack of consideration of symmetry problems probably accounts for the erroneous results obtained by earlier workers for the number of ways of randomly joining two identical chains. The total number of isomeric structures formed from the oxidation of two light and two heavy chains with 5 and 11 SH groups respectively of the human immunoglobulin GI has been found to be 4.8 X 10(16).     

Human colostrum: identification of minor proteins in the aqueous phase by proteomics

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.     

Chemical structures of three neutral oligosaccharides obtained from horse (thoroughbred) colostrum.

1. Three neutral oligosaccharides were obtained from horse colostrum by ion-exchange, activated charcoal column and preparative paper chromatographies. 2. The following structures were elucidated by methanolysis, methylation analysis and 75 MHz 13C-NMR spectroscopy; Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (HM-3a), Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc (HM-3b) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (HM-5). 3. HM-3a and HM-5 have been found in human milk, named as lacto-N-neotetraose and lacto-N-neohexaose, respectively. HM-3b has been isolated from goat milk. 4. An homology and heterogeneity were assumed among the following animal species' milk oligosaccharides: horse, human, goat and tammar wallaby.     

Peptidome analysis of human skim milk in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. Due to the difficulties associated with detection technology of the peptides, the expression of the peptides present in human milk is not known widely. In recent years, peptidome analysis has received increasing attention. In this report, the analysis of endogenous peptides in human milk was done by mass spectrometry. A method was also developed by our researchers, which can be used in the extraction of peptide from human milk. Analysis of the extracts by LC–MS/MS resulted in the detection of 1000–3000 Da peptide-like features. Out of these, 419 peptides were identified by MS/MS. The identified peptides were found to originate from 34 proteins, of which several have been reported. Analysis of the peptides’ cleavage sites showed that the peptides are cleaved with regulations. This may reflect the protease activity and distribution in human body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery. Isotope dimethyl labeling analysis was also used to test the effects of premature delivery on milk protein composition in this study. Differences in peptides expression between breast milk in term milk (38–41 weeks gestation) and preterm milk (28–32 weeks gestation) were investigated in this study. 41 Peptides in these two groups were found expressed differently. 23 Peptides were present at higher levels in preterm milk, and 18 were present at higher levels in term milk.     

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.