Location and characterization of the three carbohydrate prosthetic groups of human protein HC.

Three different carbohydrate prosthetic groups associated to three chymotryptic peptides, Q1, Q2 and Q3, were isolated from the reduced and carboxymethylated human protein HC. The first oligosaccharide forms an O-glycosidic linkage with a threonine residue at position 5 in the polypeptide chain of protein HC. The second and third carbohydrate prosthetic groups form N-linkages with asparagine residues at positions 17 and 96. Oligosaccharides present in Q1 contain 1 residue of NANA, 2 of GalNAc and 1 of Gal corresponding to the following structure: -O-GalNAc-GalNAc-Gal-NANA. Q2 contains 3 NANA, 9 GlcNAc, 2 Gal and 3 Man, and Q3 contains 2 NANA, 5 GlcNAc, 1 Gal and 2 Man. The sugar compositions of Q2 and Q3 oligosaccharides are compatible with that of the complex kind. The amount of oligosaccharides present in Q1, Q2 and Q3 corresponded respectively to 3.0%, 12.2% and 7.3% of the weight of protein HC. No difference was found between the carbohydrate composition of urinary and plasma protein HC.     

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 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.     

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.     

zFP538, a yellow fluorescent protein from coral, belongs to the DsRed subfamily of GFP-like proteins but possesses the unexpected site of fragmentation

The yellow fluorescent protein (zFP538) from coral Zoanthus sp. belongs to a family of green fluorescent protein (GFP). Absorption and emission spectra of zFP538 show an intermediate bathochromic shift as compared with a number of recently cloned GFP-like red fluorescent and nonfluorescent chromoproteins of the DsRed subfamily. Here we report that the zFP538 chromophore is very close, if not identical, in chemical structure to that of DsRed. To gain insight into the mechanism of zFP538 fluorescence and chromophore structure and chemistry, we studied three chromophore-containing peptides isolated from enzymatic digests of zFP538. Like GFP and DsRed chromophores, these contain a p-hydroxybenzylideneimidazolinone moiety formed by Lys-66, Tyr-67, and Gly-68 of zFP538. One of the peptides studied, the hexapeptide FKYGDR derivative, is a proteolysis product of the zFP538 full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other peptides are the derivatives of the pentapeptide KYGDR resulted from the protein in which the chromophore maturation process had been completed. One of these has an oxogroup at Lys-66 C(alpha) and is a hydrolysis product of another one, with the imino group at Lys-66 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain fragmentation at a very unexpected site, the former peptide bond between Phe-65 C' and Lys-66 N(alpha). Also observed in the entire protein under mild denaturing conditions, this fragmentation is likely the feature of native zFP538 chromophore that distinguishes it chemically from the DsRed chromophore.     

Amino acid sequence of the amphiphilic phosphocarrier protein factor IIILac of the lactose-specific phosphotransferase system of Staphylococcus

The lactose-specific factor III of the phosphotransferase system of Staphylococcus aureus is an amphiphilic trimeric protein composed of identical subunits. It is hydrophilic in its unphosphorylated state and can be isolated from the cytoplasmic protein fraction. It becomes a constituent of the membrane-bound phosphotransferase complex upon phosphorylation of a single histidyl residue. The sequence of S. aureus factor IIILac was determined and revealed that the subunits consist of 103 residues corresponding to a Mr of 11 367 and of 34 101 for the native trimer: (sequence; see text) According to this sequence and previous work histidine residue 82 located in the C-terminal part of the polypeptide chain is phosphorylated at the N-3 position by phosphoenolpyruvate, enzyme I, and histidine-containing phosphocarrier protein. The N-terminal part of the protein comprising approximately one-third of the chain exhibits in vitro affinity toward membrane-bound enzyme IILac.     

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.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

The complete amino-acid sequence of dimeric beta-lactoglobulin from mouflon (Ovis ammon musimon) milk

beta-Lactoglobulin from Mouflon (Ovis ammon musimon) milk has been isolated and its complete primary structure determined. This protein has been isolated in dimeric form and has a molecular mass of 37 kDa. The amino-acid sequence has been determined by microsequencing of the native protein and the peptides were obtained after tryptic cleavage. The tryptic peptides were isolated by reversed phase high-performance liquid chromatography. The primary structure of mouflon beta-lactoglobulin shows close similarity to ruminant beta-lactoglobulins. The presence of His at position 20 indicates that this protein belongs to the B-type of dimeric ovine beta-lactoglobulins. Mouflon beta-lactoglobulin is a 162 amino acid long polypeptide chain with two disulphide bridges and one free thiol group. Structural similarities to the bilin-binding protein, BG protein from olfactory epithelium and retinol-binding protein are discussed.     

Location of the disulfide bonds in the antitumor protein neocarzinostatin.

Two disulfide bonds in the antitumor antibiotic neocarzinostatin were determined chemically. The peptic and peptic/thermolytic peptides from the native protein were isolated by gel filtration and ion-exchange chromatography followed by reverse-phase HPLC. The cystine peptides obtained were oxidized separately by performic acid treatment and further separated by HPLC into cysteic acid peptides. Sequence analyses of the isolated peptides revealed the location of the disulfide bonds at Cys37-Cys47 and Cys88-Cys93.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Neutral microprotein, a novel human plasma and urinary protein associated with a yellow-brown chromophore. Isolation from protein HC preparations and partial characterization

An apparently novel human plasma and urinary protein of low molecular weight was isolated from several highly purified preparations of protein HC by gel chromatography and high voltage electrophoresis with a yield of about 8 mg/g. The protein has a molecular weight of about 20,000, neutral electrophoretic mobility at pH 6.5 and a high content of half-cystine. It is associated with a yellow-brown chromophore like protein HC and could be demonstrated in all investigated preparations of isolated human, rabbit and guinea-pig protein HC and alpha 1-microglobulin.     

Identification of the protein HC receptor

In the present study, we demonstrate for the first time the presence of a specific receptor for protein HC on the surface of human cells using the human histiocytic lymphoma cell line U937. Cells treated for 4 days with the maturation inducer phorbol 12-myristate 13-acetate, were found to increase both the number of cells binding protein HC (76% higher than for untreated cells) and the expression of protein HC receptors. Protein HC bound to these cells in a specific and saturable manner. Scatchard analysis at 4 degrees C, using radioiodinated protein HC, indicated a single class of low-affinity receptor (Ka = 2-5 x 10(7) M-1) and 20,000-30,000 receptors per cell. Monoclonal antibodies against protein HC abrogated specific binding of this protein to U937. In contrast, monoclonal antibodies that did not react with protein HC (anti-LFA-1 alpha, anti-MO1 alpha) were without effect on the binding reaction.     

Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta

A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific protein.     

Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D9K) is unstable when stored at 4 degrees C in the absence of Ca(II). Cleavage of the polypeptide occurs producing approximately 5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an Mr 13,700, in contrast to the observed Mr 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.     

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.     

Probes of beta-galactosidase structure with antibodies. Reaction of anti-peptide antibodies against native enzyme.

Antibodies were prepared against 18 tryptic and cyanogen bromide peptides from beta-galactosidase ranging in size from 15 to 96 amino acid residues representing more than 80% of the polypeptide chain. They were tested for binding capacity and affinity toward their homologous antigens and toward the whole native protein. Nine antisera bound to beta-galactosidase; these had been raised against certain peptides from the central and carboxyl-terminal regions of the poly-peptide chain. Based on these results a preliminary model of the three-dimensional structure of the folded protein is suggested.     

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.     

Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.     

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.