Fatty acylation of murine Ia alpha, beta, and invariant chains

Labeling of murine spleen cells with [3H]palmitate followed by analysis of immunoprecipitated Ia molecules indicated that Ia alpha- and beta-chains and their associated invariant chain contain covalently bound fatty acid. This modification is present in I-A and I-E molecules and has been found in all haplotypes examined. The 3H label was not dissociated from the glycoproteins by detergents or under the denaturing conditions of SDS-polyacrylamide gel electrophoresis. The fatty acid linked to Ii is released by treatment with neutral hydroxylamine, which indicates thioester linkage. The acylation of alpha- and beta-chains appears to involve attachment of palmitoyl groups via an ester linkage sensitive to alkaline hydrolysis. The radioactive species released from the isolated chains by treating with KOH/methanol co-migrated with palmitic acid and palmitic acid methyl ester on thin-layer chromatography.     

Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus

We describe the covalent attachment of palmitate to the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site. Labeling of respiratory syncytial virus-infected Vero cells with [3H]palmitate, followed by the purification and subsequent analysis of the fusion glycoprotein in conjunction with polyacrylamide gel electrophoresis, demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein. The bound palmitate was sensitive to 1 M hydroxylamine at neutral pH. In addition, the release of palmitate label by reduction with sodium borohydride showed that the palmitate is linked to the protein through a thioester bond. Isolation of a radiolabeled peptide from a tryptic digest of the protein and subsequent amino-terminal sequence analysis revealed that the cysteine residue (amino acid residue 550) within the anchor sequence, located at the carboxyl terminus of the F1 subunit, is the covalent attachment site for palmitate.     

Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and site-directed mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.     

Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus.

The cysteine residue in the cytoplasmic domain at position 489 of the sequence of the glycoprotein (G protein) isolated from vesicular-stomatitis virions is completely blocked for carboxymethylation. After release of covalently bound fatty acids by hydroxylamine at pH 6.8, this cysteine residue could be specifically labelled by iodo[14C]acetic acid. Reaction products were analysed after specific cleavage of labelled G protein at asparagine-glycine bonds by hydroxylamine at pH 9.3, which generated a C-terminal peptide of Mr 15,300 containing only the single cysteine residue. Bromelain digestion of [3H]palmitic acid-labelled membrane fractions of vesicular-stomatitis-virus-infected baby-hamster kidney cells removed almost completely the 3H radioactivity from the cytoplasmic domain of the G protein, whereas the ectodomain was completely protected by the microsomal membrane. This result indicates that the acylation site of the G protein is exposed on the cytoplasmic side of intracellular membranes. Taken together, both biochemical techniques strongly suggest that the single cysteine-489 residue, which is located six amino acid residues distal to the putative transmembrane domain, is the acylation site. The thioester bond between palmitic acid and the G protein is quite resistant to hydroxylamine treatment (0.32 M at pH 6.8 for 1 h at 37 degrees C) compared with the reactivity of the thioester linkage in palmitoyl-CoA, which is cleaved at relatively low concentrations of hydroxylamine (0.05 M).     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Thiol-fatty acylation of the glucose transport protein of human erythrocytes

Incubation of intact human erythrocytes with [3H]palmitate labeled a protein with electrophoretic characteristics of the glucose transporter. This labeling occurred via a thioester linkage, since it was unaffected by organic solvent extraction, but was substantially removed as the hydroxamate upon treatment with neutral hydroxylamine. Immunoprecipitation of the labeled protein with a monoclonal antibody to the glucose transporter confirmed its identity.     

Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor.

We report that the cell surface glycoprotein CD4 expressed in HeLa cells can be metabolically labeled with [3H]palmitic acid. Analysis of the 3H-label after hydrolysis of the protein indicated that it was incorporated predominantly as palmitic acid. Comparison of the amount of [3H]palmitate incorporated into CD4 with that incorporated into a protein known to contain one molecule of esterified palmitate suggested that one to two molecules of palmitate were added to CD4. The fatty acid was readily cleaved from CD4 by treatment with weak base suggesting a thioester linkage. Mutations of each of 2 cysteine residues, Cys394 and Cys397, in CD4 at the junction of the transmembrane and cytoplasmic domains reduced labeling with [3H]palmitic acid, and mutation of both cysteines eliminated labeling. These results indicate that both cysteines are esterified to palmitate. Modification with palmitate was not required for expression of CD4 on the cell surface or for binding of p56lck to its cytoplasmic domain.     

Localization of the human complement component C3 binding site on the IgG heavy chain

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.     

Molecular organization of the DQ subregion (DO-DX-DV-DQ) of the human MHC and its evolutionary implications

An overlapping set of cosmid clones from the homozygous DR7 B lymphoblastoid cell line MANN linking the HLA-DO through -DQ subregions is described. This region encompasses 280 kb of DNA, including DQ alpha, DQ beta, DX alpha, DX beta, DO beta, and recently indentified L chain sequences termed DV beta. The orientation and grouping of the alpha- and beta-chains is comparable with an analogous murine class II subregion and also with the HLA-DR alpha and -DR beta chains, suggesting that the arrangement of the constituent genes of class II subregions predates the mouse/human divergence.     

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol- anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.     

S acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine

S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.     

HLA-DR7-Specific monoclonal antibodies and a chimpanzee anti-DR7 serum detect different epitopes on the same molecule

We describe here two monoclonal antibodies with HLA-DR7 serologic specificity. The antibodies, SFR16-DR7M, a cytotoxic rat IgM antibody of high affinity, and SFR16-DR7G, a noncytotoxic antibody of the rat IgG 2a class, react with only DR7-positive cells in radioimmunoassay. The cytotoxic activity of SFR16-DR7M correlates completely with the presence of the DR7 specificity, and segregates with the DR7-bearing haplotype in a family. SFR16-DR7M precipitates a class II molecule with the electrophoretic characteristics of DR molecules from LG-10, an HLA-DR7 homozygous cell line. SFR16-DR7G completely inhibits the cytotoxicity of SFR16-DR7M, but only partially inhibits the cytotoxicity of a chimpanzee antiserum with DR7 specificity, Gay/Swei. In binding-inhibition studies, binding of SFR16-DR7M to LG-10 cells is only partially inhibited by the chimpanzee antiserum and vice versa. Both SFR16-DR7M and Gay/Swei reciprocally deplete the same class II molecules from a ³⁵S-methionine-labeled detergent-solubilized membrane preparation of the LG-10 cell line. The chimpanzee serum Gay contains antibodies reactive with epitopes on separated DR7 beta chains, while both SFR16-DR7M and SFR16-DR7G bind only to DR7 alpha-beta complexes. These data suggest that at least two allogeneic epitopes exist which result in the same serologic specificity, and that these epitopes differ in their requirement for alpha-beta complex formation.     

2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage

2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis.     

The reaction of LipB, the octanoyl-[acyl carrier protein]:protein N-octanoyltransferase of lipoic acid synthesis, proceeds through an acyl-enzyme intermediate

The lipB gene of Escherichia coli encodes an enzyme (LipB) that transfers the octanoyl moiety of octanoyl-acyl carrier protein (octanoyl-ACP) to the lipoyl domains of the 2-oxo acid dehydrogenases and the H subunit of glycine cleavage enzyme. We report that the LipB reaction proceeds through an acyl-enzyme intermediate in which the octanoyl moiety forms a thioester bond with the thiol of residue C169. The intermediate was catalytically competent in that the octanoyl group of the purified octanoylated LipB was transferred either to an 87-residue lipoyl domain derived from E. coli pyruvate dehydrogenase or to ACP (in the reversal of the physiological reaction). The octanoylated LipB linkage was cleaved by thiol reagents and by neutral hydroxylamine, strongly suggesting a thioester bond. Separation and mass spectral analyses of the peptides of the unmodified and octanoylated proteins showed that each of the assigned peptides of the two proteins had identical masses, indicating that none of these peptides were octanoylated. However, the one major peptide that we failed to recover was that predicted to contain all three LipB cysteine residues. These three cysteine residues were therefore targeted for site-directed mutagenesis and only C169 was found to be essential for LipB function in vivo. The C169S protein had no detectable activity whereas the C169A protein retained trace activity. Surprisingly, both proteins lacking C169 formed an octanoyl-LipB species, although neither was catalytically competent. The octanoyl-LipB species formed by the C169S protein was resistant to neutral hydroxylamine treatment, consistent with formation of an ester linkage to the serine hydroxyl group. The octanoyl-C169A LipB species was probably acylated at C147. LipB species that lacked all three cysteine residues also formed a catalytically incompetent octanoyl adduct, indicating the presence of a reactive side chain other than a cysteine thiol that lies adjacent to the active site.     

Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method

Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.     

Identification of the MT3 molecule using two-dimensional gel electrophoresis and alloantisera

The MT3 antigen is defined serologically as a DR supertypic specificity and is strongly associated with DR4, DR7, and DRw9. To determine whether the MT3 molecule is distinct from the DR molecule, DR4 and MT3 antigens were immunoprecipitated from 125I-labeled plasma membrane glycoproteins of a DR4-homozygous, MT3-homozygous B lymphoid cell line, Wa, and compared by two-dimensional (2-D) gel electrophoresis. The precipitates with two different anti-DR4 alloantisera and with three different mouse antibodies against human Ia monomorphic determinants gave the same 2-D gel pattern consisting of one heavy chain with a molecular weight of 34 000 and a set of light chains with a molecular weight of 30 000, indicating that these polypeptides are the components of the DR4 molecule. On the other hand, all three anti-MT3 alloantisera used precipitated an identical set of anti-MT3 alloantisera specific light chains with a molecular weight of 30 000, and one heavy chain with a molecular weight of 34 000. The pI of the MT3 light chain was more acidic than that of the DR4 light chain. The amount of MT3 light chains was much smaller than that of DR4 light chains in unlabeled plasma membrane glycoproteins. Thus, we have demonstrated directly using 2-D gel electrophoresis and anti-MT3 alloantisera that the MT3 antigen is a new human Ia molecule distinct from DR4.     

Acylation of disc membrane rhodopsin may be nonenzymatic

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.     

RFLP analysis of HLA-DR and -DQ genes and their linkage relationships in the Pacific

Human genomic DNA samples from Melanesians, Micronesians, and Caucasoids of known HLA-DR type were examined with cDNA probes for HLA-DR alpha, -DR beta, -DQ alpha, and -DQ beta chain genes. DR beta hybridizations with TaqI-digested DNA did not detect any new DR specificities in the Pacific. However, within the DR5 specificity a common DNA subtype was found in Pacific Islanders that was not seen in Caucasoids. Altogether, four DNA subtypes of DR5 are described. With the DQ alpha and DQ beta probes, significantly more variation could be demonstrated between populations. For example, DR2 was associated with a DQ beta TaqI pattern in the Pacific that was very rare in Caucasoids and additional RFLP analysis with other enzymes showed that this pattern is probably associated with the Dw12 subtype of DR2. DRw8-positive samples showed two different DQ alpha TaqI patterns, and these correlated with DQw1 and DQw3 specificities. DR alpha hybridizations with BglII-digested DNA also revealed different linkage relationships of the HLA-class II region genes between Pacific and Caucasoid specimens. The different population linkage disequilibrium relationships have permitted tentative assignment of TaqI fragments to either the DR beta 1 or DR beta 2 genes and are highly suggestive that the DQw1 specificity is encoded by the DQ alpha chain gene. This study shows the value of population comparisons in contributing to knowledge of the genetic organization of the genome.     

The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with [3H]palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.     

The HLA-D-associated invariant chain binds palmitic acid at the cysteine adjacent to the membrane segment

The highly polymorphic HLA-D antigens are associated with a nonpolymorphic polypeptide chain, designated invariant chain. This invariant chain is shown to incorporate fatty acid. Invariant chain metabolically labeled with [3H]palmitic acid releases its label after treatment with hydroxylamine indicating an ester linkage of the palmitic acid. The binding of fatty acid to the invariant chain inhibits the formation of S-S-linked dimers. This suggests that the sole cysteine residue of the invariant chain is blocked by binding of fatty acid. A peptide shared by [3H]palmitic acid- or [35S]cysteine-labeled invariant chain digests supports the hypothesis that the palmitic acid binds to the cysteine which is located close to the membrane-spanning domain on the cytoplasmic site. Inhibition of N-glycosylation with tunicamycin demonstrates binding of the fatty acid to the nonglycosylated precursor of the invariant chain. Additionally, blocking of fatty acylation by cerulenin inhibits further maturation of the invariant chain, as sialylation.