Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins

The human salivary acidic proline-rich proteins (aPRPs) complex was investigated by different chromatographic and mass spectrometric approaches and the main aPRPs, namely PRP-1, PRP-2 and PIF-s (15,515 amu), Db-s (17,632 amu) and Pa (15,462 amu) proteins, were detected. All these isoforms are phosphorylated at Ser-8 and Ser-22 and have a pyroglutamic moiety at the N-terminus. Apart from Pa, all the other aPRPs undergo a proteolytic cleavage at Arg-106 residue (Arg-127 in Db-s protein), that generates the small PC peptide (4371 amu) and PRP-3, PRP-4, PIF-f (11,162 amu) and Db-f (13,280 amu) proteins, all of which were detected. With regard to the Pa protein, the main form detected was the dimeric derivative (Pa 2-mer, 30,922 amu) originated by a disulfide bond involving Cys-103 residue. Besides these known isoforms, several previously undetected aPRP derivatives were found (in minor amounts): (i) the triphosphorylated derivatives of PRP-1/PRP-2/PIF-s and Db-s, showing the additional phosphate group at Ser-17; (ii) the mono-phosphorylated forms at either Ser-22 or Ser-8 of PRP-1/PRP-2/PIF-s, PRP-3/PRP-4/PIF-f, Db-s and Db-f; (iii) a nonphosphorylated form of PRP-3/PRP-4/PIF-f; (iv) the triphosphorylated and diphosphorylated forms of Pa 2-mer. Moreover, minor quantities of PRP-3/PRP-4/PIF-f lacking the C-terminal Arg (11,006 amu), and of Pa 2-mer lacking the C-terminal Gln (30,793 amu) were found. By this approach the different phenotypes of PRH1 locus in 59 different subjects were characterized.     

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.     

Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping

MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42.     

Development of diagnostics in the search for an explanation of aerotoxic syndrome

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate, an additive in engine lubricants and hydraulic fluids that is activated to the toxic 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP). Currently, there is no laboratory evidence to support intoxication of airline crew members by CBDP. Our goal was to develop methods for testing in vivo exposure by identifying and characterizing biomarkers. Mass spectrometry was used to study the reaction of CBDP with human albumin, free tyrosine, and human butyrylcholinesterase. Human albumin made a covalent bond with CBDP, adding a mass of 170 amu to Tyr411 to yield the o-cresyl phosphotyrosine derivative. Human butyrylcholinesterase made a covalent bond with CBDP on Ser198 to yield five adducts with added masses of 80, 108, 156, 170, and 186 amu. The most abundant adduct had an added mass of 80 amu from phosphate (HPO₃), a surprising result given that no pesticide or nerve agent is known to yield phosphorylated serine with an added mass of 80 amu. The next most abundant adduct had an added mass of 170 amu to form o-cresyl phosphoserine. It is concluded that toxic gases or oil mists in cabin air may form adducts on plasma butyrylcholinesterase and albumin, detectable by mass spectrometry.     

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.     

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences.     

Amino acid sequence of human myelin basic protein peptide 45-89 as determined by mass spectrometry

In order to resolve the uncertainties about the primary structure of human myelin basic protein at residues 45-89, the sequence of this peptide and its tryptic fragments were reinvestigated by fast atom bombardment mass spectrometry. The sequence at positions 77-78 was found to be His-Gly and the sequence at positions 83-84 was shown to be Glu-Asn. The Ser at position 56 was not phosphorylated, whereas the residue at position 46 or 47 showed a heterogeneity of Gly and Ser in this peptide fragment in one of two protein preparations from different patients. These results demonstrate the usefulness of fast atom bombardment mass spectrometry for primary structure information. The corrected sequence of human basic protein peptide 45-89 will permit a more detailed immunochemical analysis of this peptide and its in vivo degradation products.     

The Complete Amino Acid Sequence of Green Turtle (Chelonia mydas) Egg White Ribonuclease

Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64–65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.Data deposition: The sequence reported in this paper has been submitted to the UniProt Knowledgebase under accession No. P84844.     

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1–A6), which were confirmed by Western blotting and MALDI–TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The ¹⁹⁹Val–Ser–Tyr–Asn²⁰² sequence in peptide T12 of A1 and A2 was replaced by ¹⁹⁹Leu–Ser–His–Glu²⁰² in A3 and A4 (T12′) while in A5 and A6 this sequence was ¹⁹⁹Val–Ser–Tyr–Val²⁰² (T12″). Peptide T1 showed the presence of ¹⁰Asn in the isoforms A1–A5 while in A6 this amino acid was replaced by ¹⁰Lys (T1′). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.     

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.     

Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the Major Intrinsic Protein membrane channel family. The in vitro activity of this aquaporin is dependent on phosphorylation, and the protein is phosphorylated in vivo by a membrane-associated Ca(2+)-dependent kinase. Mutagenesis studies have implicated three serine residues as kinase targets, but only phosphorylation of Ser7 has been observed in vivo. An atomic model of PvTIP3;1 generated by homology modeling suggested that Ser7 is the only residue that would be sterically accessible to kinases. To further explain the phosphorylation of PvTIP3;1, we overexpressed this aquaporin in the methylotrophic yeast Pichia pastoris and purified the hexahistidine-tagged protein by immobilized metal affinity chromatography. Mass spectrometry confirmed that a fraction of recombinant PvTIP3;1 was phosphorylated. Phosphatase and kinase treatments indicated that Ser7 was the only residue that could be phosphorylated. In addition, mass spectrometry indicated that the native and expressed proteins are N-terminally acetylated. This is the first demonstration that a full-length, recombinant aquaporin can be produced in yeast and authentically phosphorylated in vitro. Characterization of phosphorylation-mediated gating in PvTIP3;1 will serve as a paradigm for understanding gating mechanisms of other channels.     

On the Adsorption of Human Acidic Proline-rich Proteins (PRP-1 and PRP-3) and Statherin at Solid/liquid Interfaces

The objective of the present study was to investigate the adsorption of PRP-1, PRP-3 and statherin to solid surfaces in terms of dependence on concentration, the presence of electrolyte and surface wettability. Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates of pure PRP-1, PRP-3 and statherin onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. The initial film build-up was fast and plateaus were reached within 10 min at all concentrations for both types of surfaces and all proteins. The observed adsorption and calculated diffusion rates of PRP-1, PRP-3 and statherin, respectively, indicated that the initial adsorption was mass transport controlled at low concentrations. At hydrophobic surfaces, isotherm shapes and adsorbed amounts were similar for PRP-1 and PRP-3, while statherin adsorbed to a higher extent. At hydrophilic surfaces only PRP-1 adsorbed substantially, while for PRP-3 and statherin adsorbed amounts were low. The presence of Ca 2+ ions in the phosphate buffer solution increased the adsorption of statherin and PRP-3 on hydrophobic surfaces, while PRP-1 was unaffected. On hydrophilic surfaces, all three proteins adsorbed in higher amounts in NaCl, compared to CaCl 2 at similar ionic strength. It is concluded that acidic PRPs (PRP-1 and PRP-3) and statherin readily form films on a variety of materials and solution conditions, showing that their functions may be fulfilled under a wide range of conditions.     

The glycosylation and characterization of the candidate Gc macrophage activating factor

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.     

Structural characterization and mapping of the covalently linked FAD cofactor in choline oxidase from Arthrobacter globiformis

The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carrying amino acid residue was released by acid hydrolysis. The fluorescence excitation maxima of the isolated aminoacylriboflavin, showing a hypsochromic shift of the near-ultraviolet band relative to riboflavin, and the pH-dependent flavin fluorescence confirmed the presence of an 8alpha-substituted flavin linked to histidine. Similarly, MALDI-TOF mass spectrometry showed a molecular mass corresponding to histidylriboflavin. Classical experiments used to distinguish between the N(1) and N(3) isomers all indicated that the flavin was linked to the N(1) position of the histidine residue. The position of the FAD-carrying histidine residue in the choline oxidase polypeptide was identified by tryptic cleavage of the denatured enzyme, HPLC separation of the proteolytic peptide fragments, and characterization of the purified flavin-carrying peptide by mass spectrometry and spectroscopy. The FAD moiety was assigned to the tryptic peptide, His-Ala-Arg, corresponding to residues 87-89 in the open reading frame of the previously published cDNA sequence. Further analysis of the flavopeptide by collision-induced dissociation mass spectrometry confirmed that the flavin cofactor was attached to His(87). We conclude that this variant of choline oxidase contains 8alpha-[N(1)-histidyl]FAD at position 87 in the polypeptide chain.     

Proteomics analysis of growth hormone isoforms in the human pituitary

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.     

Phospho-regulation of DDA3 function in mitosis

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue.     

Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.     

Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.