Torben E. Petersen

A thiol‐ester in α2‐macroglobulin cleaved during proteinase complex formation

The glycoprotein crs-macroglobulin ((YAM), M, 725 000, is unique among plasma proteins in being able to form complexes with proteinases from all 4 classes (EC 3.4 2 1-24) [ l-31. The function of olM is not well understood. However, the rapid clearance of osM-proteinase complexes from the blood by the reticuloendothelial system [4] and the uptake of a,M or r+M-proteinase complexes via receptors by several types of cell in culture [5-81 indicates that cuzM plays a role in the transport of proteinases and possibly other proteins into cells. The binding of proteinases to crzM is initiated by cleavage of one or more of the Mr 180 000 subunits of crzM in the ‘bait region’ 193, producing two Mr 85 000 fragments [ 1 ,I O-121. There appears to be a close-fitting binding site in this area since even auhydrotrypsin binds to crIZM [131. The exact position of cleavage in the ‘bait region’ reflects the known substrate specificity of the particular proteinase being complexed (trypsin, plasmin, thrombin’, elastase). Following cleavage, the initial complex, involving the proteinase active site and the cvzM ‘bait region’, is rearranged to form a final complex, involving a second site in the proteinase and probably also in cr2 M (L. S.-J., T. E. P., S. M., 11. Jdrnvall, in preparation). In the final complex cvzM may well be covalently linked to the proteinase [9 ,141. The subunits of ol,M can be cleaved specifically by heating, into two fragments of Mr 120 000 and Mr 60 000 [15-l 71. This polypeptide

Primary structure of the vitamin K-dependent part of prothrombin

The biosynthesis of normal prothrombin is vitamin K-dependent [ 11. During dicoumarol treatment a prothrombin is produced [2,3] which can neither be adsorbed to barium citrate nor bind calcium ions [4] and therefore is not activated in the normal Ca’+, phospholipid systems. One reason for determining the primary structure of prothrombin has been to find the structural modification caused by vitamin K. Analyses of carbohydrate and amino acid compositions as well as N-terminal sequences of normal [5-71 and dicoumarol prothrombin [8-lo] indicated no clearcut difference between the two proteins and no unusual constituents. The first clue to the identity of the structural modification was the isolation [ 1 l] of the tryptic peptides Gly-Phe-Leu-Glx-Glx-Val-Arg and Gly-Phe-Leu-Glx-Glx-Val-Arg-Lys from positions 410 and 411 in normal prothrombin. Both peptides were free of carbohydrate and carried two negative charges more at neutral pH than could be attributed to normal glutamic acid residues [ 12, 131. The adsorption of a different tryptic peptide to barium citrate [ 141 indicated that this peptide had also been modified by vitamin K. Stenflo [ 151 has recently confirmed that peptides 410 and 411 from normal prothrombin carry the extra negative charges, and found that the corresponding sequences in dicoumarol prothrombin contain normal Glu-

Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications

OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.

Purification and characterization of three proteins isolated from the proteose peptone fraction of bovine milk.

Three major proteins from the proteose peptone of bovine milk were purified by Sephadex G-75 gel chromatography, Q-Sepharose ion-exchange and additional Sephadex G-75 gel chromatography in the presence of urea. From their mobility in a gradient SDS-PAGE, the proteins were found to have molecular masses of 17, 28 and 60 kDa. The N-terminal amino acid sequence of the 17 kDa protein was found to be homologous with a camel whey protein. This protein has not previously been described in bovine milk. From the SDS-PAGE results, the 28 kDa protein was judged to be the major protein of proteose peptone, contributing approximately 25% of the total. The N-terminal amino acid sequence showed no homology to any known protein sequence, but the amino acid composition indicated that the 28 kDa protein is identical with the PP3 component from the proteose peptone fraction of bovine milk, or part of it. The 60 kDa protein was found to be bovine osteopontin, a very highly phosphorylated protein with an Arg-Gly-Asp sequence which mediates cell attachment.

Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties

Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains ∼21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function.

Amino acid sequence of the factor XIIIa acceptor site in bovine plasma fibronectin

Blood coagulation factor XIII is a proenzyme which can be activated by thrombin [I] to the transglutaminase factor XIII, [2%4]. Factor XIII, catalyzes the formation of e(y-glutamyl)lysyl amide bonds between pairs of y-chains in aggregated fibrin, resulting in its transformation to a highly stable and insoluble covalently cross-linked clot (reviewed in [S-7]). Two other plasma proteins cYz-macroglobulin and fibronectin contain acceptor sites for factor XIII, as shown by incorporation of dansylcadaverine [8]. Only fibronectin, but not aa-macroglobulin, was shown to be crosslinked to fibrin [8]. Cross-linking of fibronectin to collagen [9 ,I 0] and to StaphyZoccocus aureus cells [ 1 l] has been demonstrated. It has also been suggested that rYa-antiplasmin could be covalently linked to. fibrin in a Ca’+dependent reaction probably catalyzed by factor XIII, [ 121. Here we report the ammo acid sequence in bovine fibronectin which contains the glutamine residue labelled with radioactive putrescine by factor XIII,. This glutamine is located at position 3 from the N-terminus of fibronectin.

Primary structure of the ‘bait’ region for proteinases in α2-macroglobulin: Nature of the complex

Most proteinase inhibitors in blood plasma, e.g., antithromb~.III, Q i-antitrypsin, inter+trypsin inhibitor, are specific towards serine proteinases and form 1 :l complexes engaging the active site and thus completely inhibiting the proteinase activity. a,-Macroglobulin is a tetrameric glycoprote~,~r 725 000, consisting of four app~ently identical chains. ‘Halfmolecules’,Mr 360 000 can be obtained under nonreductive denaturing conditions [ 1,2]. Unlike the other inhibitors (YAM can form complexes with proteinases from different classes having different substrate specificities [3-51. furthermore, the active site of the complexed proteinase is accessible to smaller molecules as shown by the facts that the cr,M-trypsin can cleave synthetic substrates [6] and be inhibited by the bovine pancreatic trypsin inhibitor (Kunitz) (M, 6514) [7]. It. IS not accessible to larger protein substrates or ~hibitors, e.g., soy bean trypsin inhibitor, Mr 20 095 [?I. However, neither zymogens such

Bovine milk procathepsin D and cathepsin D: coagulation and milk protein degradation.

Cathepsin D is an indigenous aspartic proteinase in bovine milk. By competitive enzyme-linked immunosorbent assay the amount of immunoreactive cathepsin D and procathepsin D in bovine skim milk was estimated to be 0.4 microgram/ml. Immunoreactive cathepsin D purified from whey consisted of a small fraction of mature cathepsin D, but the major form was the proenzyme procathepsin D. A preparation of bovine milk procathepsin D was, like mature cathepsin D, able to degrade purified alpha s1-, alpha s2-, beta- and kappa-casein and alpha-lactalbumin, while beta-lactoglobulin was resistant to cleavage. The cleavage sites in these proteins were determined and compared with those of chymosin. Cathepsin D was capable of generating the alpha s1-I, beta-I, beta-II and beta-III fragments originally described from the action of chymosin on the respective caseins, and these fragments were subjected to further proteolysis. Cathepsin D was also able to liberate the caseinomacropeptide from purified kappa-casein, and to coagulate bovine skim milk. This demonstrated that milk contains an indigenous coagulation enzyme present mainly in the whey fraction.

Phosphorylation, glycosylation and amino acid sequence of component PP3 from the proteose peptone fraction of bovine milk

Component PP3 is a phosphorylated glycoprotein with an apparent molecular mass of 28 kDa isolated from the proteose peptone fraction of bovine milk. The function of the protein is not known. The primary structure has been determined and shown to contain 135 amino acid residues (EMBL accession no. P80195). It was phosphorylated at Ser29, Ser34, Ser38, Ser40 and Ser46. Two O-linked carbohydrate groups were found at Thr16 and Thr86, while one N-linked carbohydrate group was present at Asn77. Thr16 was only approximately 50% glycosylated. The amino sugar detected by the amino acid analyser at Thr86 was mainly galactosamine but a small amount of glucosamine was also present. The amino sugars found in the carbohydrate group linked to Asn77 were both glucosamine and galactosamine. A fragment of PP3 has been isolated from milk and shown to correspond to residues 54-135. This fragment was probably generated by plasmin hydrolysing the Arg53-Ser54 bond.

Trypsin‐induced activation of the thiol esters in α2‐macroglobulin generates a short‐lived intermediate (‘nascent’ α2M) that can react rapidly to incorporate not only methylamine or putrescine but also proteins lacking proteinase activity

The Glx-residue is located 469 residues from the C-terminus of at,M [3]. Complement component C3 also contains a reactive thiol ester [4,5], located in an identical heptapeptide sequence in itsor’chain [6]_ Fo~owinglimited proteolysis in the ‘bait’ region of cwzM [2,3] or at that site in C3 which generates C3a and C3b [4-61 the thiol esters are rapidly cleaved resulting in the appearance of free sul~yd~l groups 12-61. Reaction of azM and C3 with methylamine leads to relatively slow cleavage of their thiol esters [2,4-61. During this process a stoichiometric amount of CHsNH2 is incorporated into the reactive Glx-residue to form y-glutamylmethylamide [l-4,6,7]. As a result of this reaction the proteinase binding capacity of cu,M [ 1,2,8] and the hemolytic activity of C3