A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62, and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated.Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).

Horse ribonuclease was purified after acid extraction of pancreas by ammonium sulfate fractionation and chromatography on CM-cellulose. It is a glycoprotein with an average molecular weight of about 18 000. The pure enzyme was chromatographically and electrophoretically heterogeneous due to the heterogeneity of the carbohydrate moiety.The amino-acid sequence was determined from four series of peptides obtained by different cleavage methods. All but four peptide bonds were overlapped by one or more peptides. The polypeptide chain contains 125 amino-acid residues and carries oligosaccharide side-chains at positions 21, 34, and 62; part of Asn-21 occurs in the carbohydrate-free form. There are two additional amino acids at the C-terminus and a deletion at position 39. Including these, horse ribonuclease differs in 35 positions from the bovine enzyme. Horse ribonuclease is more closely related to the artiodactyl ribonucleases than is the rat enzyme.Except for the deletion, all differences can be accommodated in the three-dimensional model of bovine ribonuclease-S without altering the folding of the backbone. Model building of the loop containing the deletion offered an explanation for the deviating titration behaviour of one of the tyrosine residues in horse ribonuclease.Compared to the large body of information obtained from bovine pancreatic ribonuclease, little attention has been paid to ribonucleases from other species. Yet, their study may yield most valuable data on structure-function relationships as well as on the phylogeny of pancreatic ribonucleases.Beintema and Gruber [2] elucidated the aminoacid sequence of rat pancreatic ribonuclease and discovered the high rate of evolution of the pancreatic ribonucleases. This makes the phylogeny of these enzymes a very useful complement to the known pedigrees of the more slowly evolving globins and cytochromes c [3] and justifiesextension of the sequence work to pancreatic ribonucleases from other ungulates and rodents [4,' 4,5].Several studies on other pancreatic ribonucleases have been published [4, Enzymes (CBN Recommendations 1972). Ribonuclease (EC 3.1.4.22) ; carboxypeptidase A (EC 3.4.12.2) ; oc-chymotrypsin (EC 3.4.21.1) ; trypsin (EC 3.4.21.4) ; thermolysin (EC 3.4.24.4). This paper describes the isolation, some properties, and the amino-acid sequence determination of horse ribonuclease. Part of the sequence data presented in this paper appeared in a preliminary form elsewhere [2,3,5,16]. MATERIALS AND METHODS Pancreatic tissue from horse (Equus eabatlus L.)was obtained fresh from the slaughter-house. Ribosomal RNA from rooster liver was prepared as described previously [17].Ribonuclease A (bovine pancreas, RAF 8 KA), carboxypeptidase A (treated with diisopropylphosphofluoridate) and a-chymotrypsin (bovine pancreas, 3 x cryst.) were from Worthington Biochem. Corp.Trypsin (pig, 3 x cryst., batch UKB), trypsin (bovine pancreas, 1 x cryst, treated with N,N-dicyclohexyl carbodiimide), and trypsin (bovine pancreas, 1 x cryst., treated with ~-l-tosylamido-2-phenyl...

No abstract

Several analogues of the amino acid sequence of peptide 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and Ev-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9-21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10-18 and peptide 9-17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9-21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A1 6 belongs to group VII antibodies, which recognize residues 11 -19 of gD. In a previous study, three critical residues, Aspl3, Argl6 and Phel7, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(ll-l7)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11 -17, identified Argl6 as an essential residue and suggested that Asp13 and Phel7 are mainly involved in stabilization of the secondary structure of the peptide.Keywords: herpes simplex virus 1 ; glycoprotein D; linear epitope ; kinetics ; surface plasmon resonance.Glycoprotein D (gD) of herpes simplex virus (HSV) type 1 is an essential component of the virion envelope for the entry of HSV into mammalian cells [I] and is one of the HSV proteins on which immunological and vaccine studies have focused. Immunization of mice with gD of HSV-1 results in high neutralizing-antibody titers against HSV-1, and these mice are protected against a lethal HSV challenge [ 2 ] . mAbs directed against different regions of gD of HSV-1 have been divided into groups according to their antigenic sites [3], mAbs which recognize amino acid residues 11 -19 in the N-terminus are designated as group VII mAbs [4, 51. mAb A16 is a group VII mAb that binds a synthetic peptide consisting of amino acid residues 9-19 of gD [6, 71. Immunization with synthetic peptides comprising this region of HSV gD protected mice against a lethal challenge with HSV [8, 91. Substitution studies showed that Pro14 and Argl6 are important for binding of mAb A16 [7]. Additional information on residues that interact with mAb A16 was obtained by screening a random peptide library displayed on a filamentous bacteriophage [lo] with the antibody.Peptide sequences obtained by screening such a library resemble or mimic the epitope and are designated as mimotopes. We identified one mimotope. Its smallest synthetic version able to inhibit binding of the gD-(9-19)-peptide to mAb A16 was a ...