Isolation of human complex-forming glycoprotein, heterogeneous in charge (protein HC), and its IgA complex from plasma. Physiochemical and immunochemical properties, normal plasma concentration

Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two major protein HC components in blood plasma. The plasma concentrations of the low and high molecular weight protein HC components were measured by this method in 13 healthy Caucasians. The results for the low molecular weight protein HC were: mean, 20.3 mg/liter, S.D., 3.2 mg/liter, range, 13.6-26.0 mg/liter; and for the protein HC X IgA complex: mean, 293 mg/liter, S.D., 176 mg/liter, range, 36-620 mg/liter.     

SAXS studies of human protein HC (alpha1-microglobulin)

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (lambda=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration R(G)= 2.20 nm and D(max)=6.3 nm and the dimer by R(G)=2.99 nm and D(max)=9.5 nm.     

The molecular organization of the protein HC-IgA complex (HC-IgA)

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.     

The complete amino acids sequence of human complex-forming glycoprotein heterogeneous in charge (protein HC)

The complete amino acid sequence of human protein HC isolated from the urine of a single individual (JL) was determined. The polypeptide chain contained 181 residues, had a calculated molecular weight of 20,435 and contained 3 cysteine residues at positions 34, 70 and 167. An intrachain disulfide bridge was formed by residues 70 and 167. No variation of the amino acid sequence of protein HC was found and can therefore not explain the charge heterogeneity of protein HC in a given individual. The amino acid sequence of protein HC was almost identical to the one reported for human α₁-microglobulin but contained 14 additional residues.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Identification of retinol as one of the protein HC chromophores

Protein HC (alias alpha 1-microglobulin) contains so far unidentified yellow-brown fluorescent chromophores. Several preparations of human protein HC were extracted with hexane. Most of the extracts contained a substance which, upon reversed-phase HPLC, co-eluted with all-trans- retinol and had an absorption spectrum identical to that of retinol. The substance was also, like retinol, destroyed by exposure to ultraviolet light and acid pH. These observations strongly support the proposal that protein HC is a member of the newly defined lipocalin protein superfamily. The highest retinol-protein HC molar ratio of the investigated protein HC preparations was 1.6 x 10(-3) indicating that retinol is not the only ligand bound to protein HC. This was confirmed by comparing the absorption spectrum of protein HC before and after hexane extraction.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein

Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced from the sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6-kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitation of the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitation of the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

The complete amino acid sequence of human complex-forming glycoprotein heterogeneous in charge (protein HC) from one individual

The complete amino acid sequence of the single polypeptide chain of human complex-forming glycoprotein heterogeneous in charge (protein HC) isolated from a single individual is reported with the supporting data. The primary structure was determined by automatic degradation of the intact chain and of fragments obtained by chemical and enzymatic degradations of the native or reduced and S-carboxymethylated protein. The polypeptide chain of protein HC contained 182 amino acid residues with a calculated molecular weight of 20,621. No amino acid sequence variability was found and such variability can therefore not explain the great charge heterogeneity of protein HC in a single individual. The amino acid sequence of protein HC was nearly identical to the one reported for human alpha 1-microglobulin in a research communication but contained 15 additional residues.     

The protein HC chromophore is linked to the cysteine residue at position 34 of the polypeptide chain by a reduction-resistant bond and causes the charge heterogeneity of protein HC.

Protein HC, an extremely charge-heterogeneous lipocalin, carries a yellow-brown fluorescent chromophore of unknown structure covalently bound at an unidentified site of its polypeptide chain. Two chromophore-carrying peptides with 60% of the chromophore material (defined as material absorbing light at 330 nm) were isolated from pepsin-digested native human protein HC by reversed-phase high performance liquid chromatography. Sequence analysis of these peptides indicated that the chromophore was bound to the cysteine residue at position 34 of the protein HC polypeptide chain. Sequence analysis of a native chromophore-tripeptide complex, isolated from pronase digests of the pepsin-produced peptides, identified the sequence Thr33-X34-Pro35, corroborating the position of the chromophore linkage. Quantitative amino acid analysis of the hydrolyzed, performic acid-oxidized, chromophore-tripeptide complex demonstrated approximately equal amounts of threonine, cysteic acid, and proline in the complex. Reduction and carboxymethylation of the native chromophore-tripeptide complex did not remove the chromophore from the peptide. The absorption spectrum of the chromophore-tripeptide complex was similar to that of native protein HC, implying that all of the heterogeneity of protein HC resides in its chromophore.     

Cross-talk between cAMP and formylmet-leu-phe in human neutrophils: phosphorylation of a 52,000 molecular weight protein.

The mechanism of inhibition of neutrophil phagocytic functions by cAMP-elevating agents has not yet been clarified. In the present work, the effects of adenylate cyclase agonists on protein phosphorylation in the formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated human neutrophils were studied. Before stimulation, 32Pi-labelled cells were incubated with adenosine deaminase to remove the endogenously produced adenosine, an adenylate cyclase agonist itself. A protein of about 52,000 molecular weight was rapidly and transiently phosphorylated when neutrophils were stimulated with fMLP in the presence of isoproterenol, prostaglandin E1, histamine or 2-chloroadenosine. This phosphorylation was blocked by the antagonists of the receptors for the above-listed agents. No phosphorylation of the 52,000 molecular weight protein could be observed if either fMLP or the cAMP-elevating agent were applied alone. A calcium ionophore A23187 and dibutyryl-cAMP could replace fMLP and a cAMP-elevating agent, respectively. Phosphorylation of the 52,000 molecular weight protein was also demonstrated in cell lysates in the presence of cAMP, and in membrane preparations in the presence of the catalytic subunit of cAMP-dependent protein kinase. These data suggest that phosphorylation of the 52,000 molecular weight protein in intact cells is dependent on the cross-talk between the fMLP- and the cAMP-signalling pathways, and may thus be involved in the cAMP-regulatory mechanism.     

Detection of an immunoglobulin-like serum protein possessing a high affinity for collagen

Fibronectin isolated from bovine serum by affinity chromatography on collagen-Sepharose was found to contain a great number of concomitant proteins. Polyacrylamide gel electrophoresis of experimental samples pretreated with beta-mercaptoethanol under denaturation conditions resulted in the polypeptide fractions with Mr of 25, 54 and 82 KD, while the non-treated samples contained only one protein of non-fibronectin type (Mr = = 180-190 KD). This protein was isolated from the total preparations of collagen-binding proteins by the procedures generally employed for the isolation of purified preparations of immunoglobulins G; this protein was also isolated from purified immunoglobulins G using affinity chromatography on collagen-Sepharose. In terms of its molecular weight, subunit composition and immunological and chromatographical behaviour this protein can be related to immunoglobulins. The immunoglobulin-like protein isolated together with fibronectin revealed an affinity for denatured collagen, but not for fibronectin or Sepharose. The content of immunoglobulin with an affinity for denatured collagen in the total fraction of immunoglobulins G is 0.3-0.5%.     

Isolation and characterization of a dog serum lipoprotein having apolipoprotein A-I as its predominant protein constituent.

The serum high density lipoproteins (HDL) of normolipemic dogs (beagles) were isolated in the density range of p 1.063 to 1.21 g/ml, and characterized in terms of composition and physical properties (flotation and diffusion coefficients, partial specific volume, molecular weight, electrophoretic mobility, ultraviolet absorption, and circular dichroism). The results indicated that canine HDL is a relatively homogeneous class with a molecular weight of about 230 000 and general properties similar to those reported for human HDL. After delipidation, the resulting apolipoprotein, apo-HDL, was fractionated by Sephadex G-200 column chromatography in urea or guanidine hydrochloride solutions. About 90% of the apo-HDL consisted of a protein with a molecular weight of about 28 000, similar in amino acid composition to human apolipoprotein A-I and having the same NH2 terminus (aspartic acid) and COOH terminus (glutamine) and no carbohydrates. Two other proteins were isolated, one having an apparent mol wt of 55 000 and representing, at least in part, an aggregate of apolipoprotein A-I and the other component with a mol wt of 8000, not yet characterized. The results indicate that canine HDL, as an intact complex, has general physical properties that lie between those reported for human HDL2 and HDL3, and that it differs compositionally from the human products mainly in its predominant content of apo-A-I. These findings together with evidence for the relatively homogeneous nature of the canine HDL provide new prospects for unraveling the relationship between polypeptide composition and HDL structure.     

Isolation and properties of cathepsins A from chicken liver]

Four highly purified enzyme preparations, which belongs to the cathepsine A group in their substrate specificity, are isolated from the extract of a hen liver acetone powder. The preparations are designated as A1, A2, A3 and A4 according to their electrophoretic mobility. The A1 component is a protein with molecular weight of about 200 000, it degrades a synthetic substrate and, to a small degree, hemoglobin. This protein is suggested to be the fragment of a liver enzyme complex. The A2 component has molecular weight of about 70 000 and the highest activity. The A3 component has molecular weight 100 000 and the lowest activity. The A2 and A3 components are similar to cathepsine A from other tissues. THe A4 component is characterized by a high electrophoretic mobility, a resistance in neutral and weakly alkaline media and a low molecular weight (of about 60 000). Cu2+, Ag+ and diisopropylphosphofluoridate completely inhibited the activity of all the enzyme preparation studied. Tosyl-alpha-amino-phenylethyl-chloremethylketone inhibited the enzymes activity only by 50-70%.     

Expression and characterization of a synthetic protein C activator in Pichia pastoris

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1). In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.     

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.