An acetylated N-terminus of adenovirus type 2 hexon protein

An acetylated N-terminus of adenovirus type 2 hexon protein was characterized using radioactively labeled protein and mass spectrometry. The labeled protein, obtained by synthesis in a medium containing ¹⁴C-acetate, was digested with proteolytic enzymes. A radioactive peptide, Acetyl-Ala-Thr-Pro-Ser, recovered in good yield, was the only N-terminal structure detected in the protein.     

The acetylated NH2-terminus of CaATPase from rabbit skeletal muscle sarcoplasmic reticulum: a commom NH2-terminal acetylated methionyl sequence.

The amino-terminus of the CaATPase from rabbit skeletal muscle sarcoplasmic reticulum was obtained by treating the reduced and alkylated enzyme with pronase with subsequent isolation of the acetyl peptides by ion-exchange chromatography and electrophoresis. Two peptides, N-Acetyl-Met-Glu and N-Acetyl-Met-Glu-Ala-Ala were co-purified. For all amino-terminally acetylated methionyl peptides reported so far, penultimate residues are found to be either aspartic or glutamic acids.     

CCR2 chemokines bind selectively to acetylated heparan sulfate octasaccharides

Chemokines participate in well documented interactions with glycosaminoglycans (GAGs). Although many chemokine amino acid residues involved in binding have been identified, much less is known about the bound regions of GAG. Heparan sulfate (HS) is the predominant cell surface GAG, and its heterogeneous nature offers proteins a variety of structural motifs with which to interact. In the present study, we describe the interactions of three CC chemokines, MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, with HS-derived oligosaccharides. To this end, we generated and characterized a complex HS octasaccharide library containing 17 different octasaccharide compositions based on acetyl and sulfate group content. Electrospray ionization mass spectrometry was used to detect chemokine-HS octasaccharide complexes in the bound state, and an affinity purification protocol was used to select and identify chemokine-binding octasaccharides from the complex mixture. The results indicate that HS octasaccharide sulfation is the foremost requirement for chemokine binding. However, within octasaccharides of constant charge density, acetylation is also observed to augment binding, suggesting that there may be as yet undiscovered specificity in the chemokine-HS interaction.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Identification of glycated and acetylated lysine residues in human α2-antiplasmin

BackgroundThe post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.AimWe sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.MethodsWe analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.ResultsEleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.ConclusionHuman α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.     

Structures of N-terminally acetylated proteins

Primary structures of 250 characterized proteins with N-terminally acetylated residues were correlated with residue distributions and other data. Excluding multiple forms derived from characterized species variants, the structures represent 105 different types of acetylated proteins. Results of comparisons extend previous suggestions based on fewer structures and define relationships further. The N-terminal residue that is acetylated is of a limited type and is frequently a small residue, with a heavy over-representation of serine and alanine. However, the occurrence of methionine at the acetylated position is also high, whereas that of glycine is less frequent than previously estimated. Lysine is over-represented in the N-terminal region, as is aspartic and glutamic acids at a few positions close to the acetylated N-terminus (especially the adjacent position). Finally, distributions of branched-chain residues in the N-terminal region of acetylated proteins are altered in relation to those of proteins in general, isoleucine is over-represented, and leucine and valine are under-represented. The results suggest that alpha-amino-acetylated proteins have special residues in N-terminally non-hydrophobic structures. Data are compatible with a protective function for acetylation but do not exclude further role(s) in processing or other special functions.     

Vacuum-ultraviolet circular dichroism of acetylated glucans

We have measured the solution and film vacuum ultraviolet circular dichroism of a series of acetylated glucans containing α- and β-(1→3), (1→4), and (1→6) linkages. In addition to the 210-nm band studied previously, we observe the entire π-π* band near 190 nm; these bands are negative for all triacetates regardless of configuration and conformation. A band near 170 nm shows configurational sensitivity for (1→3)- and (1→6)-linked polysaccharides. The band is positive for both (1→4)-linked triacetates, but when cellulose triacetate is partially deacetylated, the 170-nm band becomes negative, thus making the correlation complete. The positive 170-nm band in cellulose triacetate films is more than an order of magnitude more intense than in any other case and, further, is accompanied by an equally large negative band near 153 nm, raising the possibility that the dichroism in the triacetate arises from strong excitonic interactions which are disrupted upon partial deacetylation.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.     

Why is p53 acetylated?

Recent studies suggest that acetylation of the p53 tumor suppressor protein is not important for its DNA binding activity, as was previously thought. We discuss here a number of theories as to how this modification may serve to regulate the protein's functions.     

Titration study of acetylated lysozyme.

To study the interaction between carboxyl groups and amino groups in native lysozyme [EC 3.2.1.17], and to identify the positions and the pK values of the abnormal carboxyl groups, N-acetylated lysozyme was prepared. The acetylation did not affect the molecular shape of the enzyme, but changed six amino groups to a non-ionizable form, leaving one amino group free; this was determined to be Lys 33. In addition, pH titration of the acetylated lysozyme in 0.2 or 0.02 M KCl aqueous solution indicated fewer titratable groups with pK(int) of 7.8 or 10.4 compared with the native protein, though the number of titratable carboxyl groups was not affected by the acetylation. From the pH titration results and structural considerations, the unititratable carboxyl groups were suggested to be Asp 48, Asp 66, and Asp 87. On the other hand, spectrophotometric titration in 0.2 M KCl showed that all three tyrosine residues are titratable in the acetylated protein, although an abnormal tyrosine residue exists in the native state. Tyr 20 was suggested to be untitratable in the pH range of 8-12.6.     

Dipeptide synthesis using acetylated trypsin derivative

A modified trypsin (AA-trypsin, acetylated with acetic acid N-hydroxysuccinimide ester) gave increased yields of Bzl-Arg-Leu-NH₂ dipeptide (90% versus 59% for native trypsin) when used in 95% acetonitrile. AA-Trypsin had decreased K_m and increased k_cat values for amide and ester substrates. k_cat/K_m also increased for each substrate upon modification. AA-Trypsin showed enhanced esterase activity in hydrophilic solvents compared with native enzyme.