Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

The structure of immunoglobulin variable regions in the horned shark,Heterodontus francisci

The heavy and light chains of pooled antibodies of the hybodont shark,Heterodontus francisci (horned shark), were subjected to amino acid sequence analysis. Yield determinations showed that more than 90% of the available polypeptides in the respective pools were sequenced. The heavy chains were homogeneous in the initial framework segment and showed a sequence homology of approximately 70% with the corresponding region of the more recently evolved nurse shark and a 45% homology with a human myeloma heavy chain. The light chains were less homogeneous and not identifiable as either kappa or lambda chains as known in higher species. The first half-cystine characteristics of the variable domain intrachain disulfide bridge of immunoglobulins was present in the same position (22 for heavy chains; 23 for light chains) in the horned shark as in mammalian species. The sequence analysis also suggested the presence of a hypervariable region in the horned shark light chains. The combined data imply that the antigen-binding function of immunoglobulins is mediated in much the same manner in this primitive shark as in more recently evolved species, including mammals.     

Intermolecular complexes between three human CD1 molecules on normal thymus cells

The first cluster of differentiation (CD1) defines at least three distinct human thymic cell-surface differentiation antigens-CD1a, CD1b, and CD1c. We looked for structural homology of the three CD1 heavy chains at their peptide level by two-dimensional peptide maps. We show here that the CD1a M _r 49 000 heavy chain and the CD1b M _r 45 000 heavy chain appear to be more homologous to each other than to the CD1c M _r 43 000 heavy chain and that only one tyrosil peptide is common to the three heavy chains. Study of the CD1 heavy chains from several individuals reveals a very limited polymorphism of these molecules. We also demonstrate here that CD1a or CD1a-like molecules and other CD1 molecules can form intermolecular complexes on the surface of normal thymus cells. Molecules that are structurally very similar to CD1a molecules are associated noncovalently either with CD1c molecules or with CD1b molecules, and only CD 1a molecules can associate covalently with CD8 molecules. In contrast, we could not find these intermolecular complexes on the surface of leukemic T-cell lines in culture.Abbreviations used in this paper CD cluster of differentiation - mAb monoclonal antibody - MHC major histocompatibility complex - NP-40 Nonidet P 40 - B2m beta-2 microglobulin - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TL thymus leukemia     

Biochemical complexity of serum HLA class I molecules

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around M _r 44 000, 40 000, and 35 000–37 000. HLA-A24-positive individuals are distinguished by higher serum levels of M _r 44 000 and 40 000 class I-like molecules than those found in HLA-A24-negative individuals. The M _r 44 000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The M _r 35 000 and 37 000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum M _r 44 000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The M _r 40 000 molecules do not contain a TM region. M _r 40 000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.Abbreviations used in this paper B2m beta-2 microglobulin - BSA bovine serum albumin - EBV-BLCL Epstein-Barr virus-transformed B lymphoblastoid cell line - mAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Human pre-α-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain

Pre-α-inhibitor (PαI) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing PαI. Then, taking advantage of the differential affinity of the members of the inter-α-inhibitor family (IαI) for heparin-Sepharose and blue-Sepharose, we isolated PαI. Its specific antitryptic activity was 580 IU/g, higher than that of IαI: 420 IU/g. Its M_r, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130 000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from PαI by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the H1 and H2 heavy chains of IαI. In reducing conditions, it was quite similar to that of H2 (M_r 85 000) but clearly different from that of H1 (M_r 78 000). Thus, the so-determined apparent M_r of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74 100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69 454) are really occupied by two N-glycans, probably of biantennary type.     

An acidic modification of the cytoplasmic domain contributes to the charge heterogeneity of the MHC class I antigens

 Polypeptide phosphorylation and sialylation of the glycan moieties contribute to the charge heterogeneity of the class I major histocompatibility complex glycoproteins. The present study demonstrates that a unique acidic modification unrelated to phosphorylation or glycosylation also affects the charge heterogeneity of the H2-K^k heavy chain of BW5147 lymphoma cells. In vitro cultivation of BW5147 cells results in changes in charge heterogeneity of the H2-K^k heavy chains due to the unique acidic modification. Sequential papain digestion of the 45 000 M _r H2-K^k glycoprotein yields a 42 500 M _r glycopolypeptide initially, followed by production of a 39 000 M _r glycopolypeptide. Results from experiments designed to localize and characterize the novel acidic modification suggest that the modification resides in the segment of the H2-K^k polypeptide located between the two papain cleavage sites. This portion of the polypeptide consists of the transmembrane region and part of the cytoplasmic domain of the H2-K^k heavy chain. At steady state, 25% of the total cell surface H2-K^k possesses this modification. In addition, the modification is mutually exclusive with the phosphorylation of the H2-K^k heavy chain at Ser-333. The possible biological significance of the novel modification of class I antigens is discussed. Received: 27 May 1997 / Revised: 10 September 1997     

A human homolog of the mouse CD8 molecule,Lyt-3: genomic sequence and expression

The CD8 antigen is a marker for those cytotoxic T cells that recognize antigen in the context of class I major histocompatibility antigens (MHC) and has now been identified in many species. In rodents the CD8 antigen is a heterodimer of two distinct chains, Lyt-2 and Lyt-3 in the mouse and OX-8 M _r 32 000 and 37 000 chains in the rat. Human CD8 has consistently been described as a homodimer/homomultimer on mature T cells made up of one chain homologous to the Lyt-2 and OX-8 M _r 32 000 chains. This paper identifies a human equivalent of the second rodent CD8 chain (Lyt-3 and OX-8 M _r 37 000 chains) at the genomic level and shows that this gene is transcribed in human thymocytes and in some acute leukemic T-cell lines. The existence of a human Lyt-3 homolog raises the possibility that human CD8, like mouse CD8, may exist as a heterodimer.     

Characterization of Japanese eel immunoglobulin M and its level in serum

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by -mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml⁻¹; it amounted to 10.3% of the total serum protein.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny

The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.     

Differential expression of laminin chains in hepatic lipocytes

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B₁ and B₂ chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of ³⁵S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of M_r=380 000 was identified by immunoprecipitation. Under non-reducing conditions only one M_r=900 000 band was revealed. High levels of B₁ and B₂ mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B₂ chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the M_r=380 000 polypeptide could be unrelated to the A chain of laminin.     

Determination of the distribution of constituent disaccharide units within the chain near the linkage region of shark-cartilage chondroitin sulfate C

A method for analyzing the distribution of constituent disaccharide units within the chain near the linkage region of chondroitin sulfate has been developed. The method consists of (a) chemical modification of the reducing terminal residue in the polysaccharide by a 2-(2,4-dinitrophenylamino)ethylamino (DNP-AEA) group, (b) controlled fragmentation of the DNP-AEA-labeled polysaccharide with chondroitinase AC-I, followed by separation of the digestion products into the DNP-AEA-labeled fragments and unlabeled fragments on octyl-Sepharose CL-4B gel, (c) fractionation of the DNP-AEA-labeled fragments into fractions having different chain-lengths of Sephadex G-100 (superfine), and (d) determination of the disaccharide unit composition of the de-dinitrophenylated products (AEA-labeled fragments) by the method combining chondroitinase AC-II treatment with HPLC analysis. A preparation of shark cartilage chondroitin sulfate C, which has been characterized well with regard to molecular species (M_r 48 000; average number of repeating disaccharide units (dp_av) 93–94; consisting of chondroitin 6-sulfated 22.5%, disulfate (D type) 10.3%, and nonsulfated units 0.4%), was analyzed by the above method. On the basis of the data obtained, distribution features of the dissacharide units within the chain near the linkage region of the polysaccharide (dp_av 27) were estimated. It was, however, difficult to propose a final primary sequence of the polysaccharide chain, although there was a definite trend towards an enrichment of 4-sulfated and nonsulfated dissacharide residues in the area close to the linkage region (dp_av 3–9 or 11). This was apparent together with an enrichment of 6-sulfated and disulfated disaccharide residues in the area distant from the linkage region (dp_av 11 of 13–27).     

Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium

 In humans, the 30 000 M _r Rhesus (Rh) polypeptide D (RhD) is a dominant antigen (Ag) of the Rh blood group system. To date, an Rh-like protein has been found in chimpanzees, gorillas, gibbons, and rhesus monkeys. Related to the 30 000 M _r Rh Ag protein are two polypeptides of 50 000 M _r , the human 50 000 M _r Rh Ag and the RhD-like protein from Caenorhabditis elegans. The function of all these proteins is not sufficiently known. Here we characterize a cDNA clone (GCRH) encoding a putative 57 000 M _r polypeptide from the marine sponge Geodia cydonium, which shares sequence similarity both to the RhD Ag and the Rh50 glycoprotein. The sponge Rh-like protein comprises 523 aa residues; hydropathy analysis hints at the presence of ten transmembrane domains. An N-terminal hydrophobic cleavage signal sequence is missing, suggesting that the first membrane-spanning domain of the sponge Rh-like protein acts as a signal-anchor sequence. The sponge Rh-like protein, like the human Rh50, lacks the CLP motif which is characteristic of the 30 000 M _r RhD. In addition, the hydropathy profile of the sponge Rh-like protein is of a similar size and shape as that of human Rh50. This data indicates that the RhD and its structurally related Rh50 glycoprotein, which are highly immunogenic in humans, share a common ancestral molecule with the G. cydonium Rh-like protein. Received: 9 April 1997 / Revised: 29 May 1997     

Two mRNAs with different 3′ ends encode membrane-bound and secreted forms of immunoglobulin μ chain

During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The μ chains of membrane and secreted IgM, μ_m and μ_s, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that μ_m and μ_s heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of μ cDNA clones from a myeloma tumor that produces both types of μ chain indicate that the μ_m and μ_s mRNAs are identical throughout the coding region up to the 3′ end of the fourth constant region (C_μ4) domain, but differ in their C terminal coding and 3′ untranslated segments. From the nucleotide sequence of the μ_m cDNA clone, we predict the amino acid sequence of the 41-residue μ_m C terminal segment or “M” (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the μ_s chain has a 20-residue hydrophilic C terminal segment after the C_μ4 domain, and the μ_m chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.     

Structure and expression of the mouse homologue of the XK gene

 The human Kx blood group antigen is carried by a 37 000 M _r apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43 000 M _r Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140 000 M _r species was detected instead of the 43 000 M _r protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93 000 M _r. Received: 22 February 1999 / Revised: 8 June 1999     

Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta

Laminins assemble into trimers composed of α, β, and γ chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin α1 and α5 chains. Comparative pulse–chase experiments and deglycosylation studies in JAR cells established that the M_r 360,000 laminin α1 chain is glycosylated into a mature M_r 400,000 band while the M_r 370,000 laminin α5 chain is glycosylated into a M_r 390,000 form that upon secretion is further processed into a M_r 380,000 form. Hence, despite the shorter peptide length of α1 chain in comparison with the α5 chain, secreted α1 assumes a larger size in SDS–PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin α1 and laminin α5 chains in laminin-1 and laminin-10. In placenta laminin α1 chain (M_r 400,000) and laminin α5 chain (M_r 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin α1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin β2 chains. Surprisingly, a fraction of the laminin α1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin β1–β3 chains, possibly pointing to an unexpected complexity in the chain composition of α1-containing laminin isoforms.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

 Interleukin 16 (IL-16) is synthesized as a 67 000 M _r precursor (pro-IL-16), but only a carboxy terminal part of 12 000–14 000 M _r is secreted by CD8(⁺) lymphocytes. This lymphokine binds to CD4 and has been shown to induce migration, affect the activation state of T cells, and inhibit immunodeficiency virus replication. It has been suggested that CD8(⁺) cell-derived soluble factors play a pivotal role in protecting natural-host nonhuman primates from developing immunodeficiency following SIV infection. In a first attempt to address this question, we cloned and sequenced the IL-16 cDNA from different primates. Here we report the pro-IL-16 sequence from chimpanzees, African green monkeys (AGM), rhesus macaques, and cynomolgus macaques. In order to compare and analyze structural motifs possibly involved in processing, intracellular targeting, or secretion, we extended our study to the New World monkeys saimiri and aotus and to the mouse. Alignments of deduced amino acids reveal that the human protein shares 99% similarity to that of chimpanzees, approximately 95% to rhesus, cynomolgus and AGM, about 90% to aotus and saimiri, and 77.5% to the mouse. Phylogenetic analyses revealed the expected evolutionary groupings. Received: 27 August 1997 / Revised: 7 October 1997     

Catfish Thrombocytes Express an Integrin-like CD41/CD61 Complex

A thrombocyte-specific antigen was identified in two closely related catfish,Ictalurus punctatusandIctalurus furcatus,by monoclonal antibodies 4-20 and 7-2. The antibodies immunoprecipitate two noncovalently associated glycoprotein chains ofM_r180,000 andM_r95,000. Under reducing conditions theM_r180,000 chain is resolved intoM_r150,000 and 32,000 subcomponents. Analysis of N-terminal amino acid sequences indicates homology of theM_r95,000 chain with the β₃integrin subunit and homology of theM_r150,000 chain with the α_IIbintegrin subunit. These antibodies induce catfish thrombocyte aggregation and alteration of cell shape. The data indicate conservation of the megakaryocyte/platelet-restricted CD41/CD61 complex in bony fish.     

Expression of Rhizobium meliloti genes for nitrogen fixation in Escherichia coli minicells: Mapping of the subunit of the nitrogenase reductase

An EcoRI fragment of Rhizobium meliloti M2011 which shows homology to Klebsiella pneumoniae DNA carrying nifH and nifD was cloned in both orientations into the Cm gene of plasmid pACYC184 and expressed in Escherichia coli minicells. Fragment specific polypeptides of M_r 12 500, 21 000, 30 000, and 31 000 could be identified. By transposon mutagenesis it was shown that two of them (M_r 12 500 and 21 000) are fusion products with parts of the chloramphenicol acetyltransferase. The other two polypeptides are specified by one coding region which could be mapped by transposon mutagenesis. There are several reasons (homology to Klebsiella nifH, sequence data and molecular weight of the gene products) to assume that this coding region represents the R. meliloti nifH gene (gene for the subunit of the R. meliloti nitrogenase reductase, RmII).     

The extra segments of sequence in rat leucocyte common antigen (L-CA) are derived by alternative splicing of only three exons and show extensive O-linked glycosylationt

The leucocyte common antigen (L-CA, CD45, or T200) consists of a family of heavily glycosylated glycoproteins of apparent M _r 180 000–240 000 found at the surface of leucocytes but not other cell types. Populations of lymphocytes express forms that differ in antigenicity, apparent M _r, and glycosylation. Some of this heterogeneity is due to polypeptide differences caused by the insertion of up to three different segments of sequence near the NH₂-terminus. We report the complete sequence of the region of the rat L-CA gene encoding the extra segments. Analysis of this sequence showed that each segment was encoded by an exon but no further exons could be identified, implying that the polypeptide heterogeneity is solely due to selection from these three exons by alternative splicing. Amino acid sequencing of glycopeptides prepared from the largest forms of L-CA indicated extensive O-linked glycosylation in at least one of the extra segments.