Invariant chain influences post-translational processing of HLA-DR molecules

HLA class II MHC molecule alpha- and beta-chains are normally synthesized in the presence of a third molecule, the invariant chain (Ii). Although Ii is not required for surface expression of HLA class II molecules, the influence of Ii on post-translational processing and maturation HLA class II molecules has not been thoroughly studied. In the present study, BALB/c 3T3 cells were transfected with HLA-DR alpha- and beta-chains with or without co-transfection with human Ii. Although Ii had no effect on the surface expression of DR, Ii did have a profound effect on the post-translational processing of both the alpha- and beta-chains. In the absence of Ii, the major species of alpha- and beta-chains were of lower m.w. than when expressed in the presence of Ii. The differences in m.w. were shown to be caused by differences in glycosylation with the majority of alpha- and beta-chains remaining unprocessed and endo H sensitive in the absence of Ii. The small proportion of alpha-chains that were processed in the absence of Ii showed an altered m.w. and altered sensitivity to treatment with endo H relative to alpha-chains processed in the presence of Ii. Pulse/chase studies demonstrated that although the majority of the alpha- and beta-chains remained unprocessed in the absence of Ii, the small amount that was processed was done so at a rate similar to that observed for alpha- and beta-chains processed in the presence of Ii. These studies demonstrate that Ii influences the post-translational processing of human class II molecules by affecting the proportion of alpha- and beta-chains that are processed and by determining the degree of processing of oligosaccharides on mature alpha-chains.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

Carnivora: the primary structure of the giant otter (Pteronura brasiliensis, Mustelidae) hemoglobin

The hemoglobin of the Giant Otter (Pteronura brasiliensis, Carnivora) contains only one component. The complete primary structures of the alpha- and beta-chains are presented. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid- and gas-phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 18 and the beta-chains 12 exchanges compared with human alpha- and beta-chains, respectively. In the alpha-chains, two substitutions involve alpha 1/beta 1-contacts and one a heme-contact. In the beta-chains one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact are exchanged. The alpha- and beta-chains of the Giant Otter are compared to those of the Common Otter and other Carnivora hemoglobins.     

Structural analysis of the oligosaccharides of DR1 and DQw1 molecules

The major glycopeptide fractions of the alpha- and beta-chains of HLA-DR1 and DQw1 molecules were isolated on columns of immobilized concanavalin A (Con A), Lens culinaris (Lens), Ricinus communis agglutinin Type I (RCA), and leuko-phytohemagglutinin. Oligosaccharides were prepared from these fractions by enzymatic digestion with Endoglycosidases H or F and were analyzed on Bio-Gel P-6. The glycopeptides tightly bound to Con A (ConA III) were mostly associated with alpha-chains and were resolved as a single oligosaccharide peak (Kd = 0.72) on Bio-Gel P-6 after Endo H digestion. Man-5 is the minimal polymannosyl structure which can be deduced for the ConA III fractions of either DQw1 or DR1 oligosaccharides. The major component of the glycopeptides of the alpha-chains of either DR1 or DQw1 molecules which were weakly bound to Con A (ConA II fraction) did not interact with RCA before or after mild acid hydrolysis or neuraminidase treatment. This component represents a biantennary complex with neither terminal galactose nor sialic acid residues with a minimal structure terminating in N-acetyl glucosamine on the Mannose alpha 1----6 arm, referred to as GnM. The ConA II fractions, which constitute 10% of the total glycopeptides of beta-chains, are associated primarily with fucosylated, sialylated biantennary oligosaccharides not seen on the alpha-chains. The ConA I unbound fractions of either alpha- or beta-chains were mostly bound to RCA after mild acid hydrolysis, suggesting that the minimal structure was a sialylated triantennary structure. The major component associated with the beta-chains was bound to Lens such that a more definite structural assignment can be made, i.e., a triantennary structure with the Mannose on the alpha 1----6 arm substituted at C-2 and C-6. The oligosaccharides of alpha- and beta-chains were resolved as broad peaks on Bio-Gel P-6, suggesting that a mixture of tri- and tetraantennary structures with variable degrees of sialylation and galactosylation were present. The structural differences reported here between oligosaccharides of alpha- and beta-chains of DQw1 and of the two subsets of DR1 molecules could be responsible in part for the differential recognition properties expected of human class II molecules encoded by distinct loci.     

Selectivity in the modification of the alpha-amino groups of hemoglobin on reductive alkylation with aliphatic carbonyl compounds. Influence of derivatization on the polymerization of hemoglobin S

The reactivity of the alpha-amino groups of the alpha- and beta-chains of hemoglobn toward reductive alkylation using limiting concentrations of the aliphatic carbonyl compounds, acetaldehyde (ethylation), glyoxylic acid (carboxymethylation), glycolaldehyde (hydroxyethylation), glyceraldehyde (dihydroxypropylation), and dihydroxyacetone (dihydroxyisopropylation) has been investigated. Hemoglobin A reductively ethylated at the alpha-amino groups eluted on CM-52 ahead of unmodified hemoglobin A, and hemoglobin A reductively ethylated at the epsilon-amino groups. This observation is similar to that seen on hydroxyethylation and dihydroxypropylation of the alpha-amino group of hemoglobin A. The presence of the alpha-hydroxyl or the carboxyl group in the carbonyl component used in the reductive alkylation influences considerably the selectivity pattern during the derivatization. The alpha-amino groups of the alpha- and beta-chains are modified to nearly the same degree during reductive hydroxyethylation as well as during reductive dihydroxypropylation. Reductive ethylation (aldehyde lacking the alpha-hydroxyl group) exhibited a slight preferential reaction at Val-1(beta). The presence of a negatively charged carboxyl group in the carbonyl component, i.e. glyoxylic acid, made this preferential reaction at Val-1(beta) even more pronounced. When the reductive alkylation is carried out with dihydroxyacetone (a ketone instead of an aldehyde), the dihydroxyisopropylation occurred at a slower rate and exclusively at Val-1(beta). The ethylation, hydroxyethylation, carboxymethylation, and dihydroxypropylation of the alpha-amino groups of hemoglobin S increased its solubility from the value of 16 g/dl for the unmodified protein to about 25 g/dl for the modified protein. Thus, the alkyl chains on the alpha-amino groups on the polymerization have a strong inhibitory influence. In order to determine the influence of the alkyl chains at the alpha-amino groups of alpha- and beta-chains on polymerization, hybrid hemoglobin S tetramers with hydroxyethylation either at Val-1(alpha) or at Val-1(beta) have been prepared. The solubility of each hybrid is about 26 g/dl. Thus, the hydroxyethyl group either on the alpha- or the beta-chain appears to interfere with the polymerization of deoxygenated HbS to the same degree. The inhibitory influence of the hydroxyethyl chain at Val-1(alpha) on the polymerization, compared with the lack of such an influence when this alpha-amino group is modified by cyanate, suggests that a carbamoyl group on Val-1(alpha) can be accommodated in the intermolecular contact region involving this segment of the molecule without seriously perturbing the mo     

Amino-acid sequence of gayal hemoglobin (Bos gaurus frontalis, Bovidae)

The amino-acid sequences of the alpha- and beta-chains of gayal hemoglobin have been determined and compared with those of bovine and yak hemoglobins. The gayal alpha-chain differs from the alpha-chains of bovine by 3 amino-acid residues and from yak I alpha- and II alpha-hemoglobins by 4 and 2 residues, respectively. The gayal beta-chain differs from bovine beta A- and beta B-chains by 3 and 4 residues, respectively and from yak beta-chains by 2 residues.     

The primary structure of hemoglobin from goldfish (Carassius auratus)

The primary structures of the alpha- and beta-chains from goldfish hemoglobin are given. The globin chains were separated by gel filtration after air-oxidation of globin. After chemical and enzymatical cleavage of the chains, the peptides were isolated by gel filtration and ion exchange chromatography on Dowex. The fish-chains have one residue more than the human chains. The alpha-chain is acetylated at the amino-terminal residue and has no cysteine. Compared with the human chains there are 66 amino-acid differences in the alpha- and 72 in the beta-chains. The implication of these differences for the physiology of the hemoglobin molecule of goldfish is discussed.     

Carnivora: the primary structure of the common otter (Lutra lutra, Mustelidae) hemoglobin

The hemoglobin of the Common Otter (Lutra lutra, Carnivora) contains only one component. The complete primary structures of the alpha- and beta-chains are presented. They were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas-phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 18 and the beta-chains 13 substitutions compared to human alpha- and beta-chains, respectively. In the alpha-chains one heme- and two alpha 1/beta 1-contacts are exchanged. In the beta-chains the replacements involve one heme-, one alpha 1/beta 1-, and one alpha 1/beta 2-contact. The alpha- and beta-chains of the Common Otter are compared to those of other Carnivora hemoglobins. The unexpected low number of substitutions between Common Otter hemoglobin and that of Lesser Panda as well as of Harbor Seal is discussed.     

Characterization of hemoglobin from the lizard Uromastix hardwickii

Hemoglobin from the tropic lizard Uromastix hardwickii was isolated. Chain separations were studied, and the whole carboxymethylated globin was cleaved with trypsin. Peptides were pre-fractionated by exclusion chromatography and finally purified by reversed phase high-performance liquid chromatography. Amino acid sequence analysis permitted ordering of peptides in alpha- and beta-chains by homology with known structures in other hemoglobins. Results show large structural variations (about 50% homology between Uromastix and viper alpha-chains) and suggest chain heterogeneity with the presence of at least two types of both the alpha- and beta-chains in the preparations.     

The primary structure of the hemoglobin of the greylag goose (Answer anser) and the unequal evolution of the beta-chains (an experimental approach to a biochemical analysis of behaviour (author's transl)]

The primary structures of the alpha- and beta-chains from greylag goose (Anser anser) hemoglobin are given. The sequence was deduced automatically in the sequenator. They differ from chicken alpha-chains in the exchange of 30, from beta-chains in the exchange of only 8 amino acid residues, respectively. The contact points of inositol pentaphosphate with the beta-chains are identical in chicken and greylag goose. Unequal evolution of the beta-chains was found, which is published here for the first time. By comparing the sequences of chicken and greylag goose and considering paleontological data, we found the mutation rate of the alpha-chains to be normal, i.e. 6 million years/mutation. This corresponds to the values for other species. The mutation rate of beta-chains is reduced and was calculated at 25 million years/mutation. This is possibly due to a specific function of beta-chains. This paper is the basis of our attempt to explain on a molecular basis the ability of bar-headed goose (Anser indicus) to fly and breathe at high altitudes.     

The primary structure of the hemoglobin from the Australian ghost bat (Macroderma gigas, Microchiroptera).

The Australian ghost bat (Macroderma gigas, Microchiroptera) has two hemoglobin components in the ratio 3:2. They share identical beta-chains and differ by three replacements in the alpha-chains. The primary structures of all three chains are presented. They could be separated by high-performance liquid chromatography. The sequences were determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides. The two alpha-chains show 18 and 19 and the beta-chains 15 exchanges compared to human alpha- and beta-chains, respectively. The divergent evolution of Macroderma gigas and Megaderma lyra, two representatives of the family Megadermatidae, is discussed. An influence of replacements at functionally important positions on the hemoglobin oxygen affinity seems unlikely.     

The primary structure of the hemoglobin from the bat Macrotus californicus (Chiroptera)

The complete primary structure of the hemoglobin from the bat Macrotus californicus (Chiroptera) is presented. This hemoglobin consists of only one component. The alpha- and beta-chains were separated by reverse phase high performance liquid chromatography. The sequences of both chains were established by automatic Edman degradation of the chains and the tryptic peptides, as well as of the C-terminal peptide obtained by acidic hydrolysis of the Asp-Pro bond in the beta-chains using the film- and gas-phase method. The sequences are compared with human hemoglobin: 15 amino-acid substitutions are found in the alpha- and 22 in the beta-chains. A comparison with the hemoglobin of Rousettus aegyptiacus and Myotis velifer shows a closer relation to the Mega- than to the Microchiroptera.     

Implications of the invariant gamma-chain on the intracellular transport of class II histocompatibility antigens

We have examined the role of the invariant gamma-chain on the intracellular transport of human class II histocompatibility antigens. mRNA was selected by hybridization to cDNA corresponding to class II alpha-, beta-, and gamma-chains, and the obtained mRNA fractions were injected individually and in various combinations into X. laevis oocytes. Translation products were isolated after various periods of chase, and their carbohydrate moieties were analyzed to monitor the subcellular localization of polypeptide chains. A mixture of alpha-, beta-, and gamma-chains, or gamma-chains alone, were transported and glycosylated to the same extent as in a B lymphoblastoid cell line. However, although alpha- and beta-chains formed a complex in the absence of the gamma-chain, the transport of this complex was slowed down. Furthermore, the glycosylation of alpha- and beta-chains appeared incomplete. Thus, the invariant gamma-chain seems to play a crucial role for the rate of transport and glycosylation of class II alpha- and beta-chains.     

The primary structure of the hemoglobin of the electric eel (Electrophorus electricus)

The blood of the Electric Eel contains only one hemoglobin component. The primary structures of the alpha- and beta-chains are presented. These were separated by high-performance liquid chromatography, using a new kind of buffer system. The alpha-chains are acetylated, and consist of 142 residues, while the beta-chains are not blocked, and consist of 147 residues. The phylogenetic distances between these and the alpha- and beta-chains of human hemoglobin are 48 and 50% amino-acid exchanges, respectively. The relationship between primary structure and the Bohr effect and Root effect is discussed, especially the significance of the serine found in position F9 beta.     

Intrinsic oxygen affinity: the primary structure of a ruminantia hemoglobin: methionine in betaNA2 of a pecora, the Northern elk (Alces alces alces)

The primary structures of the alpha- and beta-chains of hemoglobin from the Northern Elk (Alces alces alces) have been determined. The sequence was compared with the bovine chains. The oxygen affinity regarding the primary structure of the beta-chains is discussed.     

The primary structure of the hemoglobin of the Indian false vampire (Megaderma lyra, Microchiroptera)

The hemoglobin of the Indian false vampire Megaderma lyra contains only one component. In this paper, we are presenting its primary structure. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides, as well as of the prolyl-peptides obtained by acid hydrolysis of the Asp-Pro bond in the alpha- and beta-chains. The alpha-chains show 23 and the beta-chains 20 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains, three exchanges involved alpha 1/beta 1 contacts. In the beta-chains one heme-and three alpha 1/beta 1 contacts are exchanged. The functional and systematic aspects of these replacements are discussed.     

Carnivora: the primary structure of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae)

The primary structure of the alpha- and beta-chains of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae) is described. The chains were separated directly from hemoglobin by RP-HPLC. After tryptic digestion of the chains, the peptides were separated by RP-HPLC. Amino acid sequences were determined by Edman degradation in liquid and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. Paguma and human hemoglobin differ with respect to 23 amino-acid residues. Some of these amino-acid substitutions, which occur in both the alpha- and beta-chains, occur at contact sites between the subunits, and at the binding sites of heme and of organic phosphate, as well as involving residues responsible for the alkaline Bohr effect.     

Carnivora: the primary structure of the beach marten (Martes foina, Mustelidae) hemoglobin

The primary structures of alpha- and beta-chains from the hemoglobin of the Beach Marten (Martes foina, Carnivora) are presented. The globin chains were separated on CM-cellulose in 8M urea buffer. The amino-acid sequences were established by automatic liquid- and gas-phase Edman degradation of the intact chains and the tryptic peptides from oxidized chains. Comparison of the sequences with human hemoglobin shows 21 exchanges in the alpha- and 12 in the beta-chains. The differences concerning heme and interchain contact sites as well as the substitution alpha 77 (EF6)Pro----Ala are discussed. The latter is observed for the first time in a mammalian hemoglobin. The sequences are compared with those of other Carnivora. The beta-chains of Martes foina and Pteronura brasiliensis (Giant Otter) are found to be identical, but their alpha-chains differ in 7 positions. The surprising small numbers of exchanges between the hemoglobin from Beach marten and that from Lesser and Greater Panda are discussed.     

The primary structure of electric ray hemoglobin (Torpedo marmorata). Bohr effect and phosphate interaction

The blood of the Electric Ray contains a number of hemoglobin components. The primary structures of the alpha- and beta-chains of the main components are presented. These chains were purified by high-performance liquid chromatography, using a new buffer system. The alpha-chains consist of 141 residues, and the beta-chains of 142 residues; both are unblocked. The phylogenetic distances from human alpha- and beta-chains are 55% and 64% amino-acid exchanges, respectively. The relationship between primary structure and the lack of both a Bohr effect and any effector affinity is discussed, and interpreted on a molecular level with reference to the sequence presented. For the Bohr effect, the mutation beta 89 Asp----Lys is significant, while the mutations beta 2 His----Ser, beta 82 Lys----Asn and beta 142 His----Cys are important for the lack of effector affinity.