Molecular cloning of human haptoglobin cDNA: evidence for a single mRNA coding for alpha 2 and beta chains

Human haptoglobin (Hp) is a plasma glycoprotein composed of alpha and beta polypeptide chains that are covalently associated by disulfide bonds. It had been suggested that alpha and beta polypeptides could be synthesized via a common precursor polypeptide. We report the molecular cloning of DNA complementary to human Hp mRNA. One of the clones, pULB1148, carries a full length copy coding for both alpha 2 and beta polypeptides. In vitro translation of human liver mRNA hybridizing with this cDNA gives a protein mol. wt. of 49000 daltons. The sequence of the alpha 2 beta cDNA shows the presence of a single Arg residue between Gln 142 of the alpha 2 chain and Ileu 1 of the beta chain. With a few minor exceptions, the DNA sequence fits the previously published amino acid sequences. The differences are the presence of an Asp residue at position 52 of alpha 2 instead of Asn, the existence in beta of only one Lys residue between Gly 65 and the following Gln, the presence of Ser and Cys at positions 218-219 instead of Cys-Ser, and of Asp residues at positions 205 and 235 instead of Asn.     

Isolation and characterization of bovine haptoglobin from acute phase sera

A macromolecular hemoglobin-binding protein, which was not detectable in normal bovine sera but appeared during acute phase inflammation, was purified, characterized, and designated as bovine haptoglobin (Hp). The purified protein had a molecular mass of 1,000-2,000 kDa, and was composed of two kinds of peptides, a 20-kDa peptide (alpha chain) and a 35-kDa glycopeptide (beta chain) linked by disulfide bonds. Amino acid composition and N-terminal sequence analyses revealed that both peptides were homologous to each counterpart of human Hp. Studies using some reducing reagents proved that highly polymerized Hp in serum was composed of 2-20 polymerized forms of alpha 2 beta 2 tetramer. Hp could bind one molecule of hemoglobin/alpha 2 beta 2 unit. Hp with smaller sizes obtained from native Hp by partial reduction with cysteine showed almost the same Hb-binding capacity.     

The haptoglobin-gene deletion responsible for anhaptoglobinemia.

We have found an allelic deletion of the haptoglobin (Hp) gene from an individual with anhaptoglobinemia. The Hp gene cluster consists of coding regions of the alpha chain and beta chain of the haptoglobin gene (Hp) and of the alpha chain and beta chain of the haptoglobin-related gene (Hpr), in tandem from the 5' side. Southern blot and PCR analyses have indicated that the individual with anhaptoglobinemia was homozygous for the gene deletion and that the gene deletion was included at least from the promoter region of Hp to Hpr alpha but not to Hpr beta (Hpdel). In addition, we found seven individuals with hypohaptoglobinemia in three families, and the genotypes of six of the seven individuals were found to be Hp2/Hpdel. The phenotypes and genotypes in one of these three families showed the father to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, the mother to be Hp2-1 and Hp1/Hp2, one of the two children to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, and the other child to be Hp1 and Hp1/Hpdel, showing an anomalous inheritance of Hp phenotypes in the child with Hp1. The Hp2/Hpdel individuals had an extremely low level of Hp (mean+/-SD = 0.049+/-0. 043 mg/ml; n=6), compared with the level (1.64+/-1.07 mg/ml) obtained from 52 healthy volunteers having phenotype Hp2, whereas the serum Hp level of an individual with Hp1/Hpdel was 0.50 mg/ml, which was approximately half the level of Hp in control sera from the Hp1 phenotype (1.26+/-0.33 mg/ml; n=9), showing a gene-dosage effect. The other allele (Hp2) of individuals with Hp2/Hpdel was found to have, in all exons, no mutation, by DNA sequencing. On the basis of the present study, the mechanism of anhaptoglobinemia and the mechanism of anomalous inheritance of Hp phenotypes were well explained. However, the mechanism of hypohaptoglobinemia remains unknown.     

Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain

Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp.     

Studies on the structure of haptoglobin and the haptoglobin-haemoglobin complex by spin and fluorescence labelling.

Human haptoglobin (Hp) of the 1-1 type incorporated one spin or fluorescence marker per molecule; the markers were found in the beta chain. Formation of the complex between spin-labelled Hp and haemoglobin or antibody caused conformational changes in the Hp molecular, evidenced by increased participation in the electron paramagnetic resonance spectrum of the component bound with the slowly rotating marker. From fluorescence-labelled Hp, the beta chain was isolated and cleaved by CNBr; only in one of the obtained peptides, one out of 4 histidine residues was modified with the marker.     

Determination of haptoglobin genotype in an Iranian population with idiopathic generalized epilepsy

Background:

Haptoglobin (Hp) is a plasma α₂-sialoglycoprotein that contains alpha and beta chains. It displays in three common phenotypes, Hp1-1, Hp2-1, and Hp2-2. Proteins expressed by polymorphic genes have grossly different molecular sizes resulting in different diffusion rates in the brain. Haptoglobin expressed by the Hp2-2 genotype has lower hemoglobin-binding capacity than Hp1-1 or Hp2-1 and is associated with idiopathic generalized epilepsy.

Methods:

To determine polymorphism in haptoglobin genes in patients with idiopathic generalized tonic-clonic seizures, 42 men, 42 women, and 50 controls were selected for this study. Genomic DNA was extracted from blood and studied by polymerase chain reactions (PCR).

Results:

The amplified fragments for the Hp1-1 and Hp2-2 genotypes were 1757 and 3481 base pairs (bp) respectively, and the Hp2-1 genotype had both fragments, in addition to a 349-bp fragment. The distribution of the three major Hp phenotypes in epilepsy patients was 28.6 (1-1), 38.1 (2-1), and 33.3% (2-2) in the men, and 31 (1-1), 40.5 (2-1), and 28.6% (2-2) in the women. The distribution of Hp genotypes in controls was 22 (1-1), 40 (2-1), and 38% (2-2).

Conclusion:

We show that all Hp genotypes participate in idiopathic generalized epilepsy.Key Words: Epilepsy, Haptoglobin, Iran     

Evolution of haptoglobin: comparison of complementary DNA encoding Hp alpha 1S and Hp alpha 2FS.

Haptoglobin is a transport glycoprotein which removes free hemoglobin from the circulation of vertebrates. In human populations haptoglobin is polymorphic due to three alleles, Hp alpha 1F, Hp alpha 1S and Hp alpha 2. The Hp alpha 2 allele is roughly twice the length of the Hp alpha 1 alleles and is the product of a partial gene duplication possibly resulting from an unequal crossover event in a heterozygous genotype Hp alpha 1F/Hp alpha 1S. In the study described here we compare the cDNA encoding Hp alpha 1S to that encoding Hp alpha 2FS . Both have a leader sequence followed by the genotypic alpha chain sequence, a beta sequence and an untranslated sequence in the 3' end. The cDNA encoding Hp alpha 2FS is composed of alpha 1F and alpha 1S domains differing by four nucleotide replacements. Hp alpha 1S cDNA contains the same replacement site mutations found in the alpha 1S domain of Hp alpha 2FS , indicating that this coding region has sustained few, if any, mutations since its incorporation into the Hp alpha 2FS gene.     

The amino acid sequences of the alpha and beta chains of hemoglobin from the snake, Liophis miliaris

The hemoglobin of Liophis miliaris has unusual properties. The hemoglobin is dimeric in the oxy form, and the cooperativity of O2 binding is very low, but both the Bohr effect and cooperativity are greatly enhanced in the presence of ATP (Matsuura, M. S. A., Ogo, S. H., and Focesi, A., Jr. (1987) Comp. Biochem. Physiol. 86A, 683-687). Four unique chains (2 alpha, 2 beta) can be isolated from the hemolysate. The amino acid sequences of one alpha and one beta chain have been determined in an effort to understand the functional properties. Comparison of the sequences with those of the alpha and beta chains of human Hb shows the following. (i) All 7 of the residues in the beta chain normally conserved in globins are identical to those of the human chain: Gly(B6), Phe(CD1), His(E7), Leu(F4), His(F8), Lys(H10), and Tyr(HC2), except that the distal His(E7) has been replaced by Gln in the alpha chain. (ii) All heme contact residues in the beta chain are identical with those in the human chain, but two differences are present in the alpha chain: the distal His(E7) is replaced by Gln and Met(B13) by Leu. (iii) All residues that form the binding site for organic phosphates are identical to those in human Hb. (iv) The major residues that contribute to the normal Bohr effect in human Hb, Asp-beta 94, His-beta 146, and Val-alpha 1 are conserved. (v) All beta chain residues at the alpha 1 beta 2 interface are identical with those in the human chain except two: Glu(G3)----Val and Glu(CD2)----Thr; these differences in charged residues may explain the dissociation to dimers. (vi) The 23 residues of the alpha chain in the alpha 1 beta 2 contact region are identical with those of the human chain except three: Phe(B14)----Leu, Thr(C3)----Gln and Pro(CD2)----Ser. (vii) A total of 17 differences occur at the alpha 1 beta 1 interface, 11 in the alpha chain and 6 in the beta chain.     

Structural study on the carbohydrate moiety of human placental alkaline phosphatase

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.     

Time course of intracellular associations, processing, and cleavages of Ii forms and class II major histocompatibility complex molecules

To determine how changing forms of class II major histocompatibility complex proteins and associated Ii molecules in intracellular compartments of human B lymphocytes might regulate or catalyze antigen processing or presentation, we analyzed immunoprecipitates of such molecules from subcellular fractions of [35S]methionine pulse-chase-labeled, 3-day-activated B lymphocytes after homogenization and distribution in Percoll density gradients. Two-dimensional gel electrophoresis of immunoprecipitates of subcellular fractions demonstrated: 1) progressive sialic acid addition to class II major histocompatibility complex beta chains and Ii but not to gamma 2, gamma 2', gamma 3, gamma 3' (p35), or p41 and its satellites; 2) association of p35 and p41 with class II complexes at 30-60 min after pulse labeling; 3) cleavage of an immature form of Ii without sialic acid at 15-30 min after pulse labeling to a COOH-terminal, 25,000-dalton fragment, p25, with a 60-90-min half-life; 4) the presence of Ii-related p29 at only 30-min chase times; 5) an effect of chloroquine or monensin, at maximal nontoxic doses, to increase (a) the time for associations of p35 and p41 with class II complexes and (b) the half-life of p25, which was then formed from Ii at reduced levels. In addition, while the half-lives of class II alpha and beta chains and Ii were comparable within intracellular fractions of any one density, in intracellular fractions of intermediate densities the complexes appeared to be longer lived (much greater than 6 h) than in lighter fractions (2-3-h half-lives).     

Protein degradation in the mouse blastocyst.

The degradation characteristics of 56 individual newly synthesized proteins of the Day 4 mouse blastocyst have been examined employing double isotope labeling of proteins for half-life measurement and two-dimensional electrophoresis for separation of proteins. The half-lives ranged from 1 to approximately 30 h with a mean of 12.4 h. Several proteins appeared to have half-lives greater than 30 h but decay times were insufficient to provide precise information for these proteins. The results suggest there is a tendency for proteins with acidic isoelectric points to be degraded more rapidly than basic proteins, and for high molecular weight proteins to be degraded more rapidly than low molecular weight proteins. Although the regressions of these two parameters on half-life were not significant, the direction and magnitude of the trends were similar to those previously described for liver proteins. Two specific proteins, tubulin and actin, were tentatively identified, and their half-lives determined. Tubulin had a half-life of 9.0 h. The half-lives of the provisionally identified gamma, beta, and alpha forms of actin were 2.2, 8.7, and 5.4 h respectively.     

Analysis of cloned mRNA sequences encoding subfragment 2 and part of subfragment 1 of alpha- and beta-myosin heavy chains of rabbit heart

Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.     

Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.     

Evidence of tandem repeat and extra thiol-groups resulted in the polymeric formation of bovine haptoglobin: a unique structure of Hp 2-2 phenotype

Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a alphabeta linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gel-filtration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the alpha chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine alpha chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.     

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.     

An Electrophoretically Silent Polymorphism for the Beta Chains of Rabbit Hemoglobin and Associated Polyribosome Patterns

The beta chain of rabbit (Oryctolagus caniculus) hemoglobin has previously been reported to contain a single residue of isoleucine at beta(112). We have detected other rabbits with either zero isoleucyl residues or half a residue per beta chain. This character is polymorphic and inherited as a simple mendelian autosomal codominant.-Normally the modal number of ribosomes per polyribosome is 4 to 6 in reticulocyte lysates; but incubation of rabbit reticulocytes prior to lysis with L-o-methylthreonine (OMT), an isostere of isoleucine, leads to a bimodal distribution in lysates with 2-3 and 8-12 ribosomes as modes. This alteration has been attributed to ribosomal traffic jams caused by starvation for ile-tRNA at mRNA codons corresponding to the locations of isoleucyl residues at positions alpha(10), alpha(17), alpha(55) and beta(112). We have confirmed this interpretation by incubating OMT with reticulocytes from rabbits with integral, half integral and nil values for isoleucyl residues per beta chain to show that formation of the larger clusters of polyribosomes requires that beta(112) = ile.