Complete amino acid sequence of a unique protein related to the variable domain of lambda light chain from a case with Fanconi syndrome

The complete amino acid sequence has been determined of a unique protein from a 55-years-old female with multiple myeloma associated with Fanconi syndrome. It existed in a monomer form with an apparent molecular weight of 10K daltons, and was consisted of 106 amino acid residues. The sequence was characteristic of the V-region of lambda light chains and was highly homologous with that of the first 106 residues of V lambda III subgroup. The presence of an intact light chain as well as a 13K daltons fragment, corresponding to the entire C-region, strongly suggests that the unique component is a catabolic product from the intact light chain rather than an aberrant product of synthesis.     

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.     

Bovine factor VII. Its purification and complete amino acid sequence

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).     

Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry

Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis.     

Amino acid sequence of myosin essential light chain from the scallop Aquipecten irradians

The amino acid sequence of the scallop myosin essential light chain (SELC) was determined from analysis of the intact, S-carboxymethylated protein and peptides produced by cleavage at its four methionine residues by cyanogen bromide digestion and at its six arginine residues by citraconylation and tryptic digestion. SELC contains 156 amino acid residues, including three cysteines, four tyrosines, one tryptophan, two histidines, and an unblocked amino-terminal proline. The protein has a calculated Mr of 17,616. SELC is an acidic protein, with a net charge of 18- at physiological pH. Comparative analysis reveals four homologous domains (I-IV), which arose by reduplication of a gene for a small, ancestral calcium binding protein. Each domain has a helix-loop-helix structure, with all the ligands for calcium binding located within a 12-residue segment that spans the loop and the first turn of the following helix. Potential calcium binding sequences were found in the ancestral sites III (residues 94-105) and IV (residues 132-143). Mutations in critical positions in domains I and II seem to preclude the possibility of calcium binding in the amino-terminal half of SELC. An unexpected third potential calcium binding segment (at residues 119-130, predicted to be in helical conformation) was found in domain IV. A reactive thiol group (Cys-78) that is involved in binding of regulatory light chains was tentatively located in an extended "linker region", which connects the two halves of the molecule.     

The primary structure of alpha-lactalbumin from camel milk

The primary structure of camel alpha-lactalbumin was determined by analysis of the intact protein, and of CNBr fragments and enzymatic peptides from the carboxymethylated protein chain. Results show that camel alpha-lactalbumin has 123 residues and a molecular mass of 14.6 kDa. The amino acid sequence is strictly homologous to alpha-lactalbumins characterized, but also exhibits extensive differences: 39 residues differ in relation to the bovine protein and only 35 residues are conserved among hitherto known alpha-lactalbumins with characterized structures. All residues ascribed critical structural or functional roles are strictly invariant in the camel protein.     

Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity.

The myeloma IgA protein produced by plasmacytoma XRPC-25, was isolated by affinity chromatography on dinitrophenyllysine-Sepharose. The affinity constant of the intact protein or its Fab' toward 2,4-dinitrophenyl-L-lysine (Dnp) was found to be 2.6 X 10(5) M-1. In order to prepare an Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1973), Biochemistry 12, 1130) from this protein, the heavy and light chains were separated and the light chain was digested with trypsin at pH 8.2 to yield half a light chain. This digest was reassociated with the heavy chain and the recombinant was digested with papain at pH 5.7. Fractionation of this digest on a Sephadex G-75 column and Dnp-lysine-Sepharose resulted in the isolation of an Fv fragment which possesses one binding site for Dnplysine (Ka = 2.0 X 10(5) M-1). The active Fv fragment has a molecular weight of 23,400 and is composed of two peptide chains, each having a molecular weight of approximately 12,000. The N-terminal residues of these chains are aspartic and glutamic acids, which are also N-terminal in the heavy and light chains, indicating that the Fv is composed of VL and VH.     

Structural analysis of bovine pancreatic thread protein

Pancreatic thread protein (PTP) forms double helical threads in the neutralpH range after purification, undergoing freely reversible,pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Grosset al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing >99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH₂-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca²⁺-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.     

Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.     

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.     

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.     

Primary structure of the variable region of an amyloidogenic Bence Jones protein NIG-77

The complete amino acid sequence of the variable region of a Bence Jones protein NIG-77 from an individual with myeloma-associated systemic amyloidosis has been determined. This protein represents a complete light chain consisting of 216 residues and it has a sequence characteristic of V lambda I subgroup, which is closely homologous to that of another amyloidogenic V lambda I Bence Jones protein NIG-51, differing by 20 of 111 residues (82% homology). In contrast, it differs by 29 residues (74% homology) to that of non-amyloidogenic V lambda I light chain NIG-64. This finding shows that, in accordance with our previous report(1), the V lambda I-related light chains can further be divided into two distinct subsubgroups, V lambda I-1 and V lambda I-2, and the latter property seems to be more prone in association with the amyloid process.     

The covalent structure of apolipoprotein A-I from canine high density lipoproteins

The complete amino acid sequence of apolipoprotein A-I (apo-A-I) from canine serum high density lipoproteins (HLD) has been determined by automated Edman degradation of the intact protein and proteolytic fragments derived therefrom. The major strategy involved analysis of overlapping sets of peptides generated by cleavage at lysyl residues with Myxobacter protease and by tryptic hydrolysis at arginines in the citraconylated protein derivative. Canine apo-A-I has 232 residues in its single polypeptide chain and its covalent structure is highly homologous to one of the two reported sequences for human apo-A-I. As in the case for the human apoprotein, predictive analysis of the canine apo-A-I sequence suggests that it comprises a series of amphiphilic alpha helices punctuated by a periodic array of prolyl residues. Human HDL contains a second major protein component, apolipoprotein A-II (apo-A-II) that is lacking in HDL from dog serum. The absence of apo-A-II in canine HDL raised the possibility that the apo-A-I from this source might contain within its primary structure sequences related to apo-A-II and thus perform the dual function of both proteins in one. Our analysis proves that canine apo-A-I has all of the structural features of human apo-A-I and that it is not an A-I: A-II hybrid molecule.     

Amphibian lutropin from the bullfrog Rana catesbeiana. Complete amino acid sequence of the beta subunit.

The amino acid sequence of lutropin (LH) beta subunit of an amphibian, the bullfrog Rana catesbeiana, has been determined. The primary structure was determined by sequencing the intact protein (residues 1-44) and peptides originated by cyanogen bromide cleavage and lysyl endopeptidase digestion. 12 cysteine residues are conserved in the bullfrog and mammalian LH beta subunit. One sugar-chain-binding site at Asn-8 is also conserved in the bullfrog and in all mammals except humans. This glycoprotein is composed of 112 amino acid residues with a molecular mass of 12675 Da, considering the six cystine bridges and excepting the sugar chain. The bullfrog beta subunit has approximately 50% sequence identity with that of mammals and with the fish gonadotropin beta subunit, and about 40% with bullfrog follicle-stimulating hormone beta subunit.     

Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.     

Light chain variable region sequence of rabbit antipneumococcal type III polysaccharide antibody 3368.

The amino acid sequence of the amino-terminal 111 residues (variable region) for the light chain of the homogeneous rabbit antipneumococcal type III polysaccharide antibody 3368 was determined. This sequence was obtained principally through automated Edmann degradations of the intact light chain and of peptides generated by tryptic digestion of the citraconylated light chain. With these methods only 2 mumol of purified light chain was required to determine the reported sequence. When compared with the light chains of four other antipneumococcal type III polysaccharide antibodies, the 3368 light chain exhibits a unique sequence in those segments of the variable region that contribute to formation of the antigen binding site (complementarity-determining regions) (10 or 11 residue differences in 12 positions). The 3368 light chain also demonstrates an insertion of three residues relative to the other four light chains in the complementarity-determining region at positions 89 to 98. These five light chains have greater than 80% sequence homology for the portion of the variable region which is not involved in antigen binding (framework).