Isolation of the fourth component of guinea pig complement and its single polypeptide chain precursor from plasma

Guinea pig C4 (C4^gp)4 was isolated from plasma by polyethyleneglycol-4000 (PEG-4000) precipitation, lysine-Sepharose affinity chromatography removal of plasminogen and plasmin, DEAE-Sephacel, SP-Sephadex chromatography, and Sepharose CL-6B gel filtration. Both phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetate (EDTA) were present in all Chromatographie buffers, and each C4 pooled concentrate was made 0.5 mM in DFP. The overall yield for three consecutive preparations averaged 3.1% of the initial plasma hemolytic C4 activity and the average specific activity rose to 139,223 U/mg, representing an average purification factor of 157-fold. The preparation of C4 gave a single band on Ouchterlony analysis with antiserum to guinea pig serum or to purified C4. Analytical alkaline discontinuous gel electrophoresis revealed a dominant protein band and functional C4 eluted from replicate gels in this region. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced C4 gave a mol. wt of 200,000, whereas SDS-PAGE of reduced and alkylated C4 demonstrated subunits of 93,000, 75,000 and 33,000 mol. wt as well as a small amount of non-reducible 200,000 mol. wt material. Elution of the 200,000 mol. wt material recognized by SDS-PAGE under reducing conditions revealed antigenic relatedness to either the α- or β-chain of C4, suggesting that it represents a pro-C4 single chain polypeptide.     

A monoclonal antibody that recognizes the alpha chain of HLA-DR antigens

A monoclonal antibody, HC2.1, has been generated that specifically reacts with both the denatured and the in vitro translated alpha chain of the DR antigen. Although HC2.1 antibody reacted with the alpha chain of protein immunoprecipitated by two DR-specific monoclonal antibodies, L227 and LB3.1, it did not react with the alpha chain of the DQ1 antigen immunoprecipitated by the monoclonal antibody, Genox 3.53. The isoelectric focusing pattern of the alpha chain precipitated by HC2.1 antibody was invariant across a range of DR specificities within a panel of lymphoblastoid cells. The alpha chain of DR antigen from a B cell line was purified by HC2.1-Sepharose immunoaffinity chromatography and limited amino acid sequence analysis was carried out with Staphylococcus aureus SV8 protease fragments purified by high-pressure liquid chromatography. The sequence analysis confirmed that the antigen reactive with HD2.1 antibody is encoded by the DR alpha chain gene.     

Molecular cloning, expression and characterization of the ovine IL-2R alpha chain.

Interleukin-2 (IL-2) stimulates the proliferation of activated antigen-specific T cells through its interaction with high affinity receptors. This event is largely regulated by the inducible expression of the alpha-chain (CD25) which, in combination with the beta-chain and possibly additional chains, forms the high affinity IL-2 receptor (IL-2R) complex. From a concanavalin A (Con A)-activated ovine T-cell complementary DNA (cDNA) library we have isolated two cDNA clones which together constitute a 2650 base pair (bp) messenger RNA (mRNA) species encoding the ovine IL-2R alpha chain. The nucleotide sequence has high homology with analogous cDNA from other species and predicts a mature protein of 254 amino acids. In addition to the predominate 2.6 kilobase (kb) ovine IL-2R alpha chain mRNA species. Northern blot analysis of activated T-cell RNA revealed two larger mRNA species. The ovine IL-2R alpha chain cDNA was transfected into CHO cells and low affinity binding of human recombinant IL-2 demonstrated. Polyclonal antisera generated against the transfected cells cross-reacted with Con A-activated ovine lymphocytes. In addition these antisera were used to immunoprecipitate a unique 50,000 MW protein from the transfected cells. It is likely that this protein represents the expressed ovine IL-2R alpha chain cDNA which is heavily glycosylated as distinct from the 30,869 MW primary translation product. Southern blot analysis of ovine genomic DNA suggests that the ovine IL-2R alpha chain is encoded by a single copy gene.     

Purification of Chicken C3 and a Structural and Functional Characterization

A major plasma protein from chicken, analogous to mammalian complement component C3, was purified by the removal of plasminogen, precipitation with polyethyleneglycol, and ion-exchange chromatography. Purification was guided by a rabbit antiserum specific to chicken C3. The yield of native C3 was 27%, and purity and functional activity was assessed by SDS-PAGE, immunoprecipitation techniques, and the ability of the purified C3 to restore the haemolytic activity of C3-depleted chicken serum. Monoclonal antibodies were raised against purified chicken C3. These antibodies were characterized and used to prepare an immunosorbent column to deplete chicken plasma specifically of C3. Chicken C3 has a mol.wt of 185,000-195,000 and a two-chain structure with an alpha chain (118,000) and beta chain (68,000). Complement activation leads to changes in the electrophoretic mobility of chicken C3 and to a decrease in mol.wt to 144,000 corresponding to the release of a 15,000 C3a and a 34,000 C3d/C3dg fragment. Chicken C3 exists in multiple molecular forms with pI values of 6.4-6.6. A genetic polymorphism of chicken C3 based on electrophoretic mobility has not yet been detected after analysis of more than 500 individuals. The function of chicken C3 is dependent on a reactive thioester because treatment of purified chicken C3 with methylamine causes functional inactivation of C3.     

Biochemical purification of detergent-solubilized H-2 alloantigens

A multistep Chromatographic fractionation scheme is described for purifying the H-2K^b and H-2D^b major histocompatibility antigens as isolated from the murine lymphoblastoid cell line EL4 (H-2^b haplotype). The membrane-integrated antigen molecules were solubilized with the non-ionic detergent NP-40 and were purified by gel filtration chromatography, ion exchange chromatography and affinity chromatography with lentil lectin conjugated to Sepharose. During the latter procedure, use of a linear gradient monosaccharide elution effected partial separation of the H-2K^b and H-2D^b antigens. At this stage the H-2 glycoproteins were highly purified based on several criteria. Upon polyacrylamide gel electrophoresis in SDS the major band migrates with a mol. wt of approximately 45,000 daltons corresponding to the mol. wt of antigens obtained by immunoprecipitation. Moreover, near identity of the profiles of the arginine-containing tryptic peptides from chromatographically-purified and immunoprecipitated H-2K^b preparations suggests that a high degree of homogeneity has been achieved in the Chromatographic purification. As is demonstrated, mg quantities of the H-2K^b and the H-2D^b antigens can be isolated and partially separated from each other by this purification scheme thereby opening the way for structural studies of the H-2K and H-2D molecules by a variety of biochemical methods.     

Isolation of Human IgA Utilizing Protein a Affinity Chromatography

Human serum IgA was isolated employing a system of G-200 column chromatography, anion exchange (DE-52) chromatography, and passage over protein A and anti-IgG Sepharose 4B columns. Polyacrylamide gel electrophoresis (PAGE) analysis of the isolated immunoglobulin revealed polypeptides corresponding to alpha and light immunoglobulin chains of IgA which were identified immunochemically as IgA. Ultracentrifugation studies revealed that the isolated immunoglobulin co-migrated with 7S IgG markers. This procedure may serve as an alternative to classical isolation procedures of IgA from human serum.     

Isolation of Human IgA Utilizing Protein a Affinity Chromatography

Human serum IgA was isolated employing a system of G-200 column chromatography, anion exchange (DE-52) chromatography, and passage over protein A and anti-IgG Sepharose 4B columns. Polyacrylamide gel electrophoresis (PAGE) analysis of the isolated immunoglobulin revealed polypeptides corresponding to alpha and light immunoglobulin chains of IgA which were identified immunochemically as IgA. Ultracentrifugation studies revealed that the isolated immunoglobulin co-migrated with 7S IgG markers. This procedure may serve as an alternative to classical isolation procedures of IgA from human serum.     

Purification of the fourth, second and fifth components of mouse complement.

The fourth, second and fifth components of mouse complement were purified by a combination of polyethylene glycol precipitation, ion exchange chromatography and gel filtration. The final products were homogeneous on SDS-PAGE, and the activity yields were 8.5% for C4, 32% for C2 and 40% for C5. C4 was composed of three polypeptide chains with mol. wts of 90,000, 78,000 and 32,000. C2 was composed of a single polypeptide chain with a mol. wt. of 115,000 and cleaved by C1s into two fragments with mol. wts of 80,000 and 35,000. The half life of C4b2a was 7 min and was not prolonged by the iodination of C2. C2 activity could not be measured using EAC14hu or EAC14gp cells, but measurement was possible with the use of EAC14mo cells with purified C5 components of mouse complement. C5 was composed of two polypeptide chains with mol. wts of 135,000 and 84,000. This is the first report on the purification of functionally active mouse complement components C4, C2 and C5 from plasma.     

Isolation and Characterization of TR-c, an Antigen of the Reiter Treponeme Precipitating with Antibodies in Syphilis

TR-c is a Reiter treponemal antigen that cross-reacts with an antigen in Treponema pallidum (Nichols pathogenic strain). Sera from patients with secondary syphilis contain precipitating antibodies against TR-c. The isolation of TR-c from a crude bacterial sonicate involves five fractionation steps: anion exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22. Ultrogel), and affinity chromatography respectively on phenyl-Sepharose CL 4B, iminodiacetic acid-Sepharose CL 4B. and lysine-Sepharose 4B. The purified TR-c was enriched 320 times compared with the starting material. and the recovery was 22%, TR-c was shown to be a protein, it did not bind to a series of lectins. and by gel filtration and polyacrylamide gel electrophoresis (PAGE) the mol, wt was determined to be in the range of 630, 000–730.000. It was found by SDS-PAGE to be composed of identical subunits, each having a mol. wt of 48, 000. The isolated TR-c was immunochemically pure when tested in crossed immunoelectrophoresis against polyspecific anti-Reiter Ig. The purified TR-c antigen was used for production of a monospecific rabbit antiserum. Monospecific rabbit anti-TR-c gave strong fluorescence with both the Reiter treponeme and T.pallidum .     

C1q、C1r和C1s的快速分离

建立了一种同时快速分离纯化C1q及酶原形式C1r和C1s的方法。用IgG Sepharose 4B亲和层析将C1q与C1r2 s2 分离 ,接着用DEAE Sephacel离子交换层析将C1r和C1s分离 ,用C1qMcAb Sepharose 4B亲和层析将C1q进一步纯化。在分离Clr和C1s的整个过程中加入蛋白水解抑制剂PMSF和NPGB ,并控制温度在 4℃、pH 6 1、无二价阳离子的条件下 ,得到高度纯化的C1q、C1r和C1s。     

Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anti-coagulant and anti-complementary sites on the heparin molecule

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.     

Synthesis of the mouse complement component C 4 (Ss-protein) by peritoneal macrophages: kinetics of secretion and glycosylation of the subunits

Ss antigen was partially purified from mouse EDTA plasma, and a highly specific antiserum was prepared. Peritoneal exudate cells from Ss-high mouse strains were labeled in primary tissue culture with [³⁵S]methionine. Ss protein was found to be synthesized by macrophages and to be secreted into the culture medium as shown by indirect immunoprecipitations performed with this antiserum. The unreduced precipitated Ss molecule coelectrophoresed with human C4 at an apparent mol. wt. of 200000. Upon reduction, Ss protein dissociated into three subunits α, β and γ of mol. wts. 98000, 70000 and 32000. This pattern was almost identical to the one obtained for human C4. The Ss a subunit was converted by human Cls into an α subunit of approximately 85 Kdalton. This conversion is a characteristic property of C4. A single-chain intracellular precursor for the Ss protein (pre-Ss: 200 Kdalton) was also immunoprecipitated. In pulse-chase experiments, it disappeared from the cytoplasmic extract within less than 10 h in a reciprocal manner to the appearance of extracellular mature Ss protein. No significant amounts of high-mol. wt. extracellular precursors were detected. A group of four single-chain molecules of mol. wt. between 160000 and 220 000 was immunoprecipitated from the tissue culture supernatant with anti-Ss antiserum. These molecules could not be chased into mature Ss protein. The α, α′ and β subunits of Ss and the α chain of C3 incorporated [³H]mannose and [³H]glucosamine, the Ss α and C3 β chains did not.     

A glycoprotein of molecular weight 85,000 on human cells of B-lineage: detection with a family of monoclonal antibodies

Immunization of BALB/c mice with glycoproteins purified from a detergent extract of human chronic lymphocytic leukemia (CLL) cells by affinity to Lens culinaris lectin led to the production of several monoclonal antibodies with similar reactivity. One of the antibodies, 50B4, was purified and the corresponding antigen was isolated from a B-lymphoblastoid cell line extract by affinity chromatography to the 50B4-IgG immunoadsorbent. Co-purification of the antigenic activities associated with five other monoclonal antibodies was achieved. Purified and radiolabelled 50B4 antigen could be specifically immunoprecipitated not only by 50B4 but also by the other five antibodies. SDS-PAGE analysis revealed that all antibodies precipitated the same component, a polypeptide chain of apparent mol. wt 85,000 under reducing conditions. Competitive-binding studies between the purified antibodies indicated the presence of two distinct epitopes on the antigen. The epitopes, each recognized by three different antibodies, were equally accessible on the cell surface of either a B-CLL (3 X 10(5) molecules/cell), a B-lymphoblastoid cell line (11 X 10(5) molecules/cell) or two acute lymphocytic leukemia (ALL) cell lines of pre-B phenotype (5 X 10(5) and 0.8 X 10(5) molecules/cell respectively). Although the antigens purified from the strongly positive ALL cell line gave a gel pattern identical to that of the B-lymphoblastoid cell line, the antigens purified from the B-CLL extract were resolved into two distinct glycosylated polypeptides of mol. wts 85,000 and 77,000 under reducing conditions. The distribution of the antigen(s) is not restricted to cells of the B-lineage as mature T-cells and a variety of non-hematopoietic cell types express both epitopes of the antigen(s).     

Analysis of hepatitis B surface antigen components solubilized with Triton X-100.

Three glycoproteins of intact hepatitis B surface antigen (HBsAg) with mol. wt. of 32 000, 30 000 and 28 000 respectively were identified by reaction with 125I-concanavalin A (Con A) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen was effectively disrupted with Triton X-100 to produce a structure with a sedimentation coefficient of 3.9S. Affinity chromatography of disrupted HBsAg using concanavalin A-Sepharose 4B (Con A-Sepharose) resulted in two fractions. The first contained material which did not bind to the lectin and consisted of a single polypeptide of mol. wt. 64 000. Further studies revealed this component to be serologically identical to serum albumin and to lack any affinity for antibody to HBsAg. A comparison of the tryptic peptide map of this polypeptide with that of purified serum albumin demonstrated identical amino-acid sequences. The second fraction contained material which bound to Con A and contained two polypeptides with mol. wt. of 28000 and 23000 respectively. HBsAg reactivity was associated with this fraction. This procedure allows the prepartion of HBsAg sub-units in milligram quantities for further immunological studies.     

Localization of a hydrophobic domain in human C5

The fifth component of human complement (C5), under physiological salt conditions, bound firmly to phenyl-Sepharose. This indicated the presence of hydrophobic sites on C5. These sites may play an important role in C5 haemolytic activity since this activity was inhibited by chaotropic ions (Mg2+, SCN-, I-) at relatively low concns (0.6-1 M). Charge shift experiments performed on purified C5 provided further evidence for the presence of hydrophobic sites within C5. Bidirectional charge shifts of C5 mobility were observed with the two detergent systems TX-100 + DOC and TX-100 + CTAB. In order to localize these sites, C5 was subjected to trypsin digestion. Three major fragments were evidenced by two-dimensional electrophoresis (agarose/SDS-PAGE and SDS-PAGE/SDS-PAGE), and were isolated by hydrophobic affinity chromatography. The first fragment, C5FI, was composed probably of a mixture of six alpha chain fragments (alpha 1-alpha 6) linked to the beta chain by disulphide bridges. The second fragment, C5FII, was composed of the beta chain linked to two alpha chain fragments: alpha 2 (Mr = 58 K) and alpha 4 (Mr = 32 K). The third fragment, C5FIII, contained two covalently linked chains (beta chain and alpha 4). The rank order for mol. wt, agarose gel electrophoretic mobility and hydrophobicity was respectively: C5 greater than or equal to C5FI greater than C5FII greater than C5FIII; C5 less than or equal to C5FI less than or equal to C5FII much less than C5FIII; and C5FII greater than or equal to C5 greater than C5FI much greater than C5FIII. From the relative hydrophobicity, the hydrophobic sites of C5 can be localized to the alpha 2 fragment (Mr = 58 K) since C5FIII lacked the alpha 2 fragment and was not hydrophobic. Cleavage and the liberation of the N-terminal part of C5 induced an increase in hydrophobicity of the remaining part of C5 (C5b and C5FII), suggesting a possible role of the hydrophobic sites in association of C5b with membranes.     

Purification of alpha-2 macroglobulin by immunoadsorbent chromatography.

A method for preparation of alpha-2 macroglobulin (alpha2M) by immunoadsorbent chromatography utilizing rabbit--anti-human alpha2M-conjugated Sepharose 4B is described. This procedure offers a rapid, simple and inexpensive method for purification of alpha2M from as little as 2.0 ml of human serum. By multiple applications to the absorbent column, as much as 10 mg of purified alpha2M may be easily prepared in one day. This technique is especially useful for isolation of alpha2M from individual sera and for studies of cultured cell supernatants containing human alpha2M.     

Purification and characterization of IgE produced by human myeloma cell line, U266

Human IgE was isolated for the first time from the supernatant of the culture fluid of a human myeloma cell line, U266. The purification procedure consisted of salting out from the supernatant with ammonium sulfate, affinity chromatography on a lysine-Sepharose 4B column, ion exchange chromatography on a DEAE-Sephacel column, gel filtration and recycling chromatography on a Sephacryl S-300 column and removal of bovine proteins on an anti-bovine serum rabbit IgG-Sepharose 4B column. One hundred and twenty eight milligrams of IgE was recovered from 461 of culture fluid. The purification was extremely simplified by the introduction of immunoaffinity chromatography using the monoclonal antibody prepared by immunizing a mouse with an IgE preparation obtained by the above method: about 3.3 mg was recovered from 960 ml of culture fluid. The purified preparation was homogeneous as judged by the double-immunodiffusion test and end group analysis. The amino acid and carbohydrate compositions of the preparation coincided with those reported on other preparations obtained from the sera of myeloma patients. Our preparation, however, showed two bands with apparent mol. wts of 240,000 and 230,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When it was reduced with dithiothreitol and analyzed by electrophoresis, it gave two heavy chains and one light chain with apparent mol. wts of 80,000 and 76,000, and 28,000, respectively. On the other hand, the IgE molecule that was synthesized and secreted into the medium in the presence of tunicamycin (0.5 microgram/ml) gave only one heavy chain and one light chain with apparent mol. wts of 62,000 and 28,000, respectively. These results demonstrated that the two IgE molecular species contained in our preparation differed from each other in the carbohydrate moiety in their heavy chains.     

Immunochemical characterization of human TL-like (T48) and Ly 1-like (T72) glycoproteins using two-dimensional polyacrylamide gel electrophoresis

Xenoantisera, designated AT48 and AT72, were developed by immunizing rabbits with human thymus cell membrane and guinea-pigs with a T-cell glycoprotein purified from leukaemic T-cell membrane. Whereas AT48, after appropriate absorption, reacted exclusively with the majority of thymocytes (mainly cortical thymocytes) among normal lymphoid populations, AT72 reacted with virtually all of the thymus and T cells but not with B cells. Thymocytes, which were strongly reactive with AT72, existed in the thymic medulla, but cortical cells were also very weakly reactive with AT72. When cultured T-cell lines, all of which were derived from patients with T-cell-type acute lymphatic leukaemias, were tested for their reactivities with AT48 and AT72 by immunofluorescence, we found that AT48 stained certain T-cell lines, whereas AT72 stained all of the T-cell lines tested so far. Immunochemical data showed that AT48 precipitated a 48K molecular weight (mol. wt) glycoprotein from 125I-labelled thymus cell surface glycoproteins, which appeared to be very weakly associated with a 12K mol. wt component. These 48K and 12K mol. wt components precipitated by AT48 showed almost identical isoelectric points (pI) to those of HLA heavy chain and beta 2-microglobulin respectively. AT72, on the other hand, precipitated a 72K mol. wt glycoprotein from thymus and T cells as well as from leukaemic T cells. A less prominent 65K mol. wt glycoprotein was also precipitated by AT72 from thymus and T cells but not from leukaemic T cells. These two components showed almost identical pI ranging approximately from 4 to 7, and this marked charge heterogeneity observed was reduced by neuraminidase treatment, suggesting that it reflects the heterogeneity in sialylation of this molecular species. We concluded from these data that AT48 and AT72 used in this work could detect human homologues of mouse TL and Ly 1 antigens respectively.     

Sandwich enzyme-linked immunosorbent assays for the quantification of the C4 isotypes (C4A and C4B) in human plasma

Sandwich enzyme-linked immunosorbent assays were developed to determine the concentration of the isotypes of the fourth component of human complement (C4A and C4B) in human plasma. In the case of C4A a monoclonal antibody against a common determinant of the alpha chain was used to capture the protein. The bound antigen was then detected with a biotinylated monoclonal antibody reacting exclusively with the C4A isotype, followed by peroxidase labeled avidin. For the quantification of C4B, C4B-specific monoclonal antibodies were coated onto a microtitration plate in order to capture the protein. Bound antigen was then detected with a biotinylated monoclonal antibody directed against C4 followed by peroxidase labeled avidin. The assays, which were rapid, selective and specific for C4A and C4B, respectively, provide an alternative to gel electrophoresis and blot procedures for the study of unexpressed alleles ('null alleles') at each of the C4 loci.     

Purification and physicochemical properties of C1q from guinea-pig serum

An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains.