Structural analysis of N-glycans from yellow lupin (Lupinus luteus) seed diphosphonucleotide phosphatase/phosphodiesterase.

N-linked oligosaccharide chains released by hydrazinolysis from yellow lupin seed diphosphonucleotide phosphatase/phosphodiesterase were fluorescence labeled and separated by high performance liquid chromatography (GlycoSep N and GlycoSep H columns). Exoglycosidase sequencing elucidated the structures of 24 separated N-glycans. Thirty percent of isolated glycans were found to be of high-mannose type (three to eight mannosyl residues), 42% were complex type and 26% belonged to paucimannosidic type. Among complex type glycans, structures with Lewis(a) epitope were identified. It is very unusual to find all types of plant N-glycans in one protein. Possible reasons for such a broad spectrum of N-glycan structures are discussed.

Purification and characterization of acid phosphatase from yellow lupin (Lupinus luteus) seeds.

Acid phosphatase (EC 3.1.3.2) from yellow lupin (Lupinus luteus) seeds was purified to homogeneity by ammonium sulphate fractionation, affinity chromatography, cation-exchange chromatography, gel filtration or reverse-phase HPLC. The enzyme is a dimer with the 50 kD and 44 kD subunits and contains 7.3% of carbohydrate, forming at least four oligosaccharide chains. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.28 mM and Vmax = 1780 IU/mg of protein. The purified phosphatase has the highest specific activities reported for any plant acid phosphatases measured for any native or synthetic substrate. The enzyme has broad specificity; however, cyclic nucleotides, pyrophosphate or phytate are not cleaved. It is inhibited by molybdate, fluoride and phosphate. There is no change in the enzyme activity in the presence of EDTA, phenanthroline and tartrate.

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds.

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.

Specificity of human cathepsin G.

A series of tetrapeptide p-nitroanilide substrates of the general formula: suc-Ala-Ala-Pro-Aaa-p-nitroanilide was used to map the S1 binding pocket of human cathepsin G. Based on the kcat/Km parameter, the following order of preference was found: Lys=Phe>Arg=Leu>Met>Nle=Nva>Ala>Asp. Thus, the enzyme exhibits clear dual and equal trypsin- and chymotrypsin-like specificities. Particularly deleterious were beta-branched side chains of Ile and Val. The P1 substrate preferences found for cathepsin G are distinctly different from many other serine proteinases, including fiddler crab collagenase and chymotrypsin. The kcat/Km values obtained for P1 Lys, Phe, Arg and Leu substrates correlate well with those determined for analogous P1 mutants of basic pancreatic trypsin inhibitor (BPTI) obtained through recombinant techniques. To characterise the subsite specificity of the enzyme, a series of Cucurbita maxima trypsin inhibitor I (CMTI I) mutants were used comprising P2-P3' and P12' positions. All the mutants obtained were inhibitors of cathepsin G with association constants in the range: 105-109 M-1. Some of the mutations destabilised complex formation. In particular, Met8-->Arg substitution at P3', which increased association constant for chymotrypsin 46-fold, led to a 7-fold decrease of binding with cathepsin G. In addition, mutation of Ile6 at position P1' either to Val or Asp was deleterious for cathepsin G. In two cases (Ala18-->Gly (P12') and Pro4-->Thr (P2)), about a 10-fold increase in association constants was observed.

Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases.

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP-3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and alpha-chymotrypsin destroyed the inhibitory activity of TIMP against MMP-3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.

Function of carbohydrate moiety in acid phosphatase of Rhodotorula glutinis.

The properties of native and partially deglycosylated forms of acid phosphatase from Rhodotorula glutinis were compared. The removal of carbohydrate moiety resulted in higher thermostability and resistance to proteolysis whereas specific activity, pH optimum and Km value with p-nitrophenyl phosphate remained unchanged. The role of carbohydrate moiety in stabilization of the enzyme structure and protection against proteolysis is suggested.

The use of sequential affinity chromatography for separation of human neutrophil elastase, cathepsin G and azurocidin.

Elastase, cathepsin G and azurocidin from human neutrophils are key components of body inflammatory defense. Perturbations in regulation of their activities lead to many serious pathological states. The paper describes a simple, fast and efficient method of joint purification of these proteins with the use of sequential affinity chromatography on squash trypsin inhibitor (CMTI I) and bovine pancreatic trypsin inhibitor (BPTI).

Acid phosphatase of the yeast Rhodotorula rubra. Purification and properties of the enzyme.

1. Acid phosphatase from the yeast Rhodotorula rubra was purified 44-fold. The purification procedure involved mechanical disruption of cells, precipitation with ethanol, chromatography on DEAE- and CM-cellulose. 2. The purified enzyme is homogeneous in polyacrylamide gels at pH 4.5, 9.5 and 8.4. Carbohydrate content accounts for 57% of the total weight. The optimum pH is at 4.0-4.6, and the enzyme is stable over pH range from 2.6 to 6.0. Full activity was retained on 60-min incubation at 50 degrees C, but it was reduced by half on 60-min incubation at 65 degrees C. 3. Specificity of the enzyme is fairly broad; monoesters of carbohydrates, and nucleosides and inorganic pyrophosphate can serve as substrates. Km was found to be 1 X 10(-4) M for p-nitrophenyl phosphate as a substrate. The enzyme is inhibited by molybdate, phosphate, arsenate and fluoride ions.

Neutrophil elastase and cathepsin G: structure, function, and biological control.

When neutrophils invade inflamed areas of the body to remove either dead or foreign components they inadvertently release potent enzymes which can, if not properly controlled, cause severe damage to healthy tissue. This can lead to a myriad of diseases including emphysema, rheumatoid arthritis, and glomuerlopnephritis, all of which are really problems of abnormal connective tissue turnover due to uncontrolled protelysis by neutrophil elastase and cathepsin G. An important step in elucidating the functions of both elastase and cathepsin G has been made by virtue of the fact that the amino acid sequence of each has been determined. Furthermore, the crystal structure of one, neutrophil elastase, is now understood. With this knowledge in mind and with the potential for a similar understanding of the mechanism of action of cathepsin G, it should soon be possible to produce synthetic inhibitors of each enzyme which can act as adjunct inhibitors to those naturally circulating in the blood or present in other tissues. As a result there is great hope for reducing the severity of injury produced by these enzymes and, therefore, in decreasing the risk for development of the debilitating diseases associated with abnormal proteolysis by neutrophil proteinases.

The action of neutrophil elastase on intact and oxidized (met)-enkephalin-arg6-gly7-leu8 peptide.

Neutrophil elastase has been found to cleave met-enkephalin-arg6-gly7-leu8 between met5 and arg6 thereby releasing the active opiate met-enkephalin. Oxidized met-enkephalin octapeptide is not attacked by this enzyme. These data suggest a potential role for neutrophil proteinases and oxidases in the regulation of opiate production in tissues.

Acid phosphatase released to the growth medium by Rhodotorula glutinis. Purification and some properties of the enzyme.

The purification procedure of acid phosphatase released by the cells of Rhodotorula glutinis to the growth medium is elaborated, as a result of which a homogeneous preparation of the enzyme is obtained. The properties of the enzyme isolated from the medium with the enzyme purified from the cells are compared. The differences in molecular weight, carbohydrate content, thermal and pH-stability and sensitivity towards SH-group reagents are found. Possible reasons for these differences are discussed.

Oligosaccharide and polypeptide homology of lupin (Lupinus luteus L.) acid phosphatase subunits.

Peptide mapping of lupin acid phosphatase clearly demonstrated the homology between its two subunits. Sequenced tryptic peptides also showed 78% identity (92% similarity) to the red bean acid phosphatase. Peptides exclusive for the 50-kDa subunit are homologous to N-terminally located sequences in red bean acid phosphatase, leading to the assumption that the shorter subunit of lupin acid phosphatase is generated by the deletion of the N-terminal part of the longer subunit. Carbohydrate moiety was found to be identical in both subunits. Oligosaccharide chains released by hydrazinolysis from the both subunits were fluorescently labeled and separated by HPLC. The structure of oligosaccharides was elucidated by exoglycosidase sequencing. Seventeen percent of isolated glycans were found to be of the high-mannose type, while the rest belonged to plant complex-type structures. Most of the complex glycans were fucosylated and xylosylated; some were fucosylated or xylosylated only.

The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus.

The interaction of three proteinases (seryl, cysteinyl, and metallo-) from Staphylococcus aureus with human plasma alpha 1-proteinase inhibitor has been investigated. As expected, none of the enzymes was inactivated by this protein, each, instead causing the conversion of the native inhibitor into an inactive form of decreased molecular weight. Amino-terminal sequence analysis indicated that inhibitor inactivation had occurred by peptide bond cleavage near the reactive center of this protein. When the inhibitor was modified by this treatment, it became resistant to both pH and temperature denaturation and, in contrast to the intact denatured protein, did not undergo further proteolytic degradation. This process of inactivation of alpha 1-proteinase inhibitor by pathogenic proteinases could result in a deregulation of its target enzyme, neutrophil elastase, and, therefore, may be important in the consumption of some plasma proteins by this enzyme during septicemia.

An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center.

Horse leukocyte elastase inhibitor rapidly forms stable, equimolar complexes with both human leukocyte elastase and cathepsin G, porcine pancreatic elastase, and bovine alpha-chymotrypsin. Formation of the inhibitor-pancreatic elastase complex results in peptide bond cleavage at the reactive site of the inhibitor so that a small peptide fragment representing the carboxyl-terminal sequence of the inhibitor is released. Sequence analysis of both this peptide, as well as that of an overlapping peptide obtained by enzymatic inactivation of native inhibitor with either Staphylococcus aureus metalloproteinase, Pseudomonas aeruginosa elastase, or cathepsin B, yields data which indicate that the reactive site encompasses a P1-P1' Ala-Met sequence. However, unlike the human endothelial plasminogen activator inhibitor, which also has a Met residue in the P1' position, oxidation of the horse inhibitor only slightly reduces its association rate constant with either of the elastolytic enzymes tested or with chymotrypsin. Comparison of the amino acid sequence at or near the reactive site of the horse inhibitor (P2-P18') with members of the serpin superfamily of proteinase inhibitors indicates that it not only belongs in this class but also represents the first example of a functionally active intracellular serpin.

Serpins: structure and mechanism of action.

The Serpins are a major family of proteins, most of which are involved in the regulation of proteinase activity. Current data indicate that inhibitor function is dependent on formation of tight, but reversible binary complexes, with carbohydrate being unimportant for this function. The reaction takes place in a reactive site loop common to all Serpins, with the key residue for complex formation being in the P1 reactive site position. However, other contact residues are also involved as shown by the variation in specificity in a number of animal proteins with the same P1 residue. The role of other amino acid residues in Serpins which aid in conferring specificity has not yet been established. However, the availability of methods for obtaining site specific mutations should soon make it possible to determine other contact points required for Serpin function, thus allowing for the design of inhibitors which are singularly targeted with a high reaction rate towards a given proteinase.

The isoforms of human neutrophil elastase and cathepsin G differ in their carbohydrate side chain structures.

The two proteinases found in human neutrophil granules, elastase and cathepsin G, each are normally isolated as a mixture of isoforms differing only in carbohydrate content. Elastase has two N-glycosylation sites occupied (Asn-45 and Asn-144), whereas cathepsin G has only one (Asn-64). Analysis of a minor form of elastase (E-1) and cathepsin G (C-1) indicates that the carbohydrate structures at each glycosylation site are complex-type bi-antennary chains usually associated with secretory glycoproteins. In contrast, the isoforms E-3 and C-3, the major forms of elastase and cathepsin G respectively, contain exclusively truncated, oligomannose-type chains at the same positions in the sequence of each protein. These data suggest the possibility that certain elastase and cathepsin G isoforms (E-1 and C-1) might be destined for secretory, others (E-3 and C-3) for lysosomal functions.

Factors affecting the oligomeric structure of yeast external invertase.

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

Primary structure of human neutrophil elastase.

The complete amino acid sequence of human neutrophil elastase has been determined. The protein consists of 218 amino acid residues, contains two asparagine-linked carbohydrate side chains, and is joined together by four disulfide bonds. Comparison of the sequence to other serine proteinases indicates only moderate homology with porcine pancreatic elastase (43.0%) or neutrophil cathepsin G (37.2%). In particular, many of the residues suggested to play important roles in the mechanism by which the pancreatic elastase functions are significantly changed in the neutrophil enzyme, indicating alternative types of binding with the human proteinase.