Robert L. Hill

A possible three-dimensional structure of bovine α-lactalbumin based on that of hen's egg-white lysozyme

Abstract Bovine α-lactalbumin and hen egg-white lysozyme have closely similar amino acid sequences. A model of α-lactalbumin has been constructed on the basis of the main chain conformation established for lysozyme. The side chain interactions of lysozyme are listed (Table 2) and the consequences of the side chain replacements in α-lactalbumin examined. Changes in internal side chains are generally interrelated in a convincing manner, suggesting that the model is largely correct, but there are some regions where it has not been possible to deduce the conformation unequivocally. Glu 35, which acts as a proton donor in lysozyme, is absent in α-lactalbumin, in which a neighbouring histidine residue may assume a similar function. The surface cleft, which is the site of substrate binding in lysozyme, is shorter in α-lactalbumin. While this would be consistent with α-lactalbumin having a mono- or disaccharide as substrate, the biochemical evidence shows that the role of α-lactalbumin in the synthesis of lactose is a complex one requiring direct interaction with the A protein.

The Structure and Assembly of Secreted Mucins

Mucins are major glycoprotein components of the mucous that coats the surfaces of cells lining the respiratory, digestive, and urogenital tracts, and in some amphibia, the skin. They function to protect epithelial cells from infection, dehydration, and physical or chemical injury, as well as to aid the passage of materials through a tract. Individual organisms make several structurally different mucins, and a given mucin may be found in more than one organ (see Supplemental Material). Members of the mucin family can differ considerably in size. Some are small, containing a few hundred amino acid residues, whereas others contain several thousands of residues and are among the largest known proteins. Irrespective of size, all mucin polypeptide chains have domains rich in threonine and/or serine whose hydroxyl groups are in O-glycosidic linkage with oligosaccharides. Moreover, these domains are composed of tandemly repeated sequences that vary in number, length, and amino acid sequence from one mucin to another (1). The carbohydrate content of a mucin may account for up to 90% of its weight. There are two types of mucins, membrane-bound and secreted. Of the human mucins, two are membrane-bound (MUC1 and MUC4) (2, 3) and four are secreted (MUC2, MUC5AC, MUC5B, and MUC7) (4–7). The three other mucins (MUC3, MUC6, and MUC8) (8–11) cannot be classified. Each human mucin has a counterpart in other animals. Thus, porcine submaxillary mucin (PSM) (12), one of the most thoroughly characterized mucins, has a tissue distribution and structure similar to MUC5B. An increasing number of proteins that are not mucins also contain highly O-glycosylated domains called “mucin-like domains.” The functions of mucins are dependent on their ability to form viscous solutions or gels. Although the highly glycosylated domains of mucins are devoid of secondary structures, they are long extended structures that are much less flexible than unglycosylated random coils. The oligosaccharides contribute to this stiffness in two ways, by limiting the rotation around peptide bonds and by charge repulsion among the neighboring, negatively charged oligosaccharide groups (13). Such long, extended molecules have a much greater solution volume than native or denatured proteins with little or no carbohydrate and endow aqueous mucin solutions with a high viscosity. Mucins protect against infection by microorganisms that bind cell surface carbohydrates, and mucin genes appear to be up-regulated by substances derived from bacteria, e.g. lipopolysaccharides (14). This review will summarize what is known about the polypeptide structures of the secreted mucins and how some, in particular PSM, are assembled via interchain disulfide bonds into molecules with molecular weights in the millions. We will not consider membrane-bound mucins, which were the subject of earlier reviews (1, 15, 16).

The effect of fibrin-stabilizing factor on the subunit structure of human fibrin

The formation of human fibrin from fibrinogen has been examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a method which separates a mixture of proteins on the basis of differences in molecular weight. It has been found that the plasma from a patient with a congenital deficiency of fibrin-stabilizing factor forms clots lacking the cross links among the alpha- and gammachains found in normal, cross-linked human fibrin. The addition of purified fibrin-stabilizing factor or normal plasma to the deficient plasma results in extensive cross-linking of the chains. Thus, the fibrinogen in the fibrin-stabilizing factor deficient plasma appears to be normal and forms fibrin which contains dimeric, cross-linked gamma-chains and polymeric, high molecular weight forms of alpha-chains. By the use of these electrophoretic methods, it has also been possible to develop a highly sensitive method for measuring the content of fibrin-stabilizing factor in plasma. This method depends upon the use of urea-treated fibrinogen, which is completely devoid of fibrin-stabilizing factor, but which forms the usual cross-linked subunits after conversion to fibrin by thrombin in the presence of fibrin-stabilizing factor.

The subunit polypeptides of human fibrinogen

Abstract Three polypeptide chains have been isolated from S -sulfofibrinogen by chromatography on carboxymethylcellulose. The three chains, designated A, B, and C, were pure as judged by ultracentrifugal analysis and zone electrophoresis. Each chain corresponded to one of the three electrophoretic components observed in unfractionated S -sulfofibrinogen. By the methods of sedimentation-equilibrium analysis in the ultracentrifuge, the molecular weights of the chains were estimated to be 47,000 for the A-chain, 56,000 for the B-chain, and 63,500 for the C-chain. The amino acid composition and tryptic peptide pattern of each chain was unique. If it is assumed that human fibrinogen contains one pair of each of the unique chains, then the molecular weight and amino acid composition of fibrinogen are closely accounted for by the weights and composition of the chains.

Lactose Synthetase: Progesterone Inhibition of the Induction of α-Lactalbumin

Lactose synthesis in the mammary gland is dependent on the hormonally controlled synthesis of the two protein components of lactose synthetase, α-lactalbumin and a galactosyltransferase. Prolactin induces the synthesis of both proteins in mammary gland explants treated with insulin and hydrocortisone, but the induction kinetics cannot account for the asynchronous synthesis of the two proteins that are observed in vivo. Progesterone appears to take part in the control of lactose synthesis and acts to repress the formation of α-lactalbumin throughout pregnancy. At parturition, when the concentration of progesterone in the plasma decreases, the rate of α-lactalbumin synthesis increases.

Mechanism of Ancrod Anticoagulation A DIRECT PROTEOLYTIC EFFECT ON FIBRIN

Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by beta-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the alpha-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded alpha-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.

Norrie Disease Protein (Norrin) Forms Disulfide-linked Oligomers Associated with the Extracellular Matrix

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent M r = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to ∼20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent M r = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.

Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains.

COS-7 cells expressing 1,360 residues from the amino terminus of porcine submaxillary mucin were used to determine whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel electrophoresis showed a single N-glycosylated protein with no indication of proteolytically processed forms. Without prior reduction, only two proteins, corresponding to monomeric and disulfide-linked trimeric species, were observed. The expressed protein devoid of N-linked oligosaccharides also formed trimers, but was secreted from cells in significantly less amounts than glycosylated trimers. Pulse-chase studies showed that the disulfide-linked trimers were assembled inside the cells no earlier than 30 min after protein synthesis commenced and after the intracellular precursors were N-glycosylated. Trimer formation was inhibited in cells treated with brefeldin A, monensin, chloroquine, or bafilomycin A1, although only brefeldin A prevented the secretion of the protein. These results suggest that trimerization takes place in compartments of the Golgi complex in which the vacuolar H+-ATPase maintains an acidic pH. Coexpression in the same cells of the amino-terminal region and the disulfide-rich carboxyl-terminal domain of the mucin showed that these structures were not disulfide-linked with one another. Cells expressing a DNA construct encoding a fusion protein between the amino- and carboxyl-terminal regions of the mucin secreted disulfide-linked dimeric and high molecular weight multimeric species of the recombinant mucin. The presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region of mucin to aid in the assembly of multimers is consistent with its amino acid identities to the amino-terminal region of human von Willebrand factor, which also serves to form disulfide-linked multimers of this protein.

The Complete cDNA Sequence and Structural Polymorphism of the Polypeptide Chain of Porcine Submaxillary Mucin

The complete structure of the DNA encoding the polypeptide chain of porcine submaxillary mucin has been determined. The polypeptide is composed of distinct domains. A large central domain containing tandem repeats of 81 residues each is flanked by much shorter domains with sequences similar to the tandem repeats. Four disulfide-rich domains, three at the amino terminus and one at the carboxyl terminus, complete the chain. The disulfide-rich domains have significant sequence identity to those of other mucins and prepro-von Willebrand factor. The coding region of the mucin gene is highly polymorphic, and three alleles were identified in a single animal that encoded different numbers of the 81-residue tandem repeats. A single large exon devoid of introns encodes the tandem repeat domains. The largest allele with 135 tandem repeats encoded 13,288 amino acids to give a polypeptide with M r = 1,184,106. The other two alleles contained 99 and 125 tandem repeats, respectively. Each allele also showed different restriction fragment length polymorphisms, which is consistent with the different patterns seen in individual animals. Fragment length polymorphism was also seen within two different families of animals, indicating that the polymorphism observed occurs in a single generation.

[37] Peptide mapping with automatic analyzers: Use of analyzers and other automatic equipment to monitor peptide separations by column methods

Publisher Summary This chapter discusses the use of analyzers and other automatic equipment to monitor peptide separations by column methods. The technique for peptide mapping of enzymic digests of proteins is applied to detect differences in amino acid sequence among the human abnormal hemoglobins closely related primate hemoglobins, the subunit fractions of rabbit γ-globulin, and genetically determined variants of other proteins. Mapping is useful for determining the number and kind of subunit polypeptides in multichain proteins. The most promising variation involves the use of automatic column chromatographic equipment. When peptide maps are obtained on paper, impure peptides are obtained often in low yield. Two procedures employed for mapping are discussed. The first is a technique that yields only column chromatographic peptide maps, whereas the second is a technique for obtaining the peptide map and simultaneously collecting the peptides represented by the peaks on the map. An automatic amino acid analyzer is modified so that the tryptic peptides are resolved on an ion-exchange column by gradient elution with pyridine-acetic acid buffers. The flow rate is maintained by a constant volume, positive displacement pump. The eluate from the column is analyzed in a manner essentially identical to that employed in amino acid analysis.