Ligatin: A Peripheral Membrane Protein with Covalently Bound Palmitic Acid*

The ligatin monomer is a polypeptide of M, = 10,000 which is soluble in acidified chloroform:methanol, a characteristic similar to that of Folch-Lee proteolipid. The hydrophobicity of ligatin is also reflected by its ability to interpolate into the phosphatidylcholine bi- layer as shown by a concentration-dependent change in membrane conductance. However, unlike other pro- teolipids the amino acid composition of ligatin is not enriched in hydrophobic amino acids (isoleucine, leu- cine, valine, methionine, phenylalanine, tryptophan). Instead, the hydrophobic character of ligatin could be explained, at least in part, by the covalent association of fatty acids, 1.4-1.7 mol of palmitate/10,000 g of protein, as revealed by gas chromatography mass spec- trographic analyses. The post-translational addition of fatty acid may therefore be the means by which ligatin acquires an affinity for membranes. The enterocytes neonatal is 7.5-nm square particles were are attached to a fibrillar array (ligatin). The square particles have been identified P-hexosaminidase and attachment to the filament is the formation

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as decribed by Jakoi et al. (1). Briefly, the procedure involved isolating a crude membrane pellet from homogenates of epithelial scrapings of the ileum by centrifugation a t 27,000 X g for 10 min. The crude membrane pellet was further fractionated by centrifugation through a sucrose step gradient made up of equal quantities (2 ml) of the following (w/w) percents of sucrose: 45%, 36%, sample (34%), and 26%. The gradient was overlaid with HEPES' buffer (10 mM HEPES, 1 mM NaN3, pH 7.6). The preparation was centrifuged in an SW41 rotor a t 95,000 X g for 45 min. Material at the 26/34% and 34/36% sucrose interfaces was pooled, resuspended in HEPES buffer, and centrifuged at 27,000 X g for 10 min. The membrane pellet was treated with 10 mM CaClz (pH 6.0, 5 min, 4 "C) to release a sedimentable ligatin-@-hexosaminidase complex. The treated pellet was then dialyzed against HEPES:EGTA buffer (5 mM HEPES, 0.5 mM EGTA, pH 7.6) for 16 h. After dialysis ligatin no longer sedimented a t 110,000 X g. Solubilized components were removed from membranes by centrifugation at 110,000 X g for 90 min and then fractionated by sieve chromatography on a Bio-Rad P-100 column (1.5 X 30 cm, 4 "C) equilibrated in HEPES buffer. The fractions containing ligatin were pooled and filtered through a PM-10 (Amicon) filter. Thirty pg of ligatin were routinely isolated from 50 animals. The purified ligatin was next extracted with organic solvents in order to remove noncovalently associated lipids. The protein was suspended in 0.5 ml of water and extracted with 1.5 ml of chloroform:methanol (l:l, v/v), to which 0.5 ml of chloroform was added to generate a two-phase system. The sample was centrifuged a t 1000 X g for 10 min. The organic phase was removed, dried under nitrogen (Nz) and found to lack fatty acids by gas chromatographic analysis. The aqueous layer containing the ligatin polypeptide was retained and lyophilized. Since only complexed polyphosphoinositides (PIP and PIP2) would not have been removed under these conditions, the lyophilized sample was applied to a neomycin affinity column prepared by reductively coupling neomycin to glycophase-CPG as described by Schacht (5). Prior to application of the sample, the neomycin column (0. 5  were dried under Nz, resuspended in 10 pl of hexane, and l-2-~1 aliquots were subjected to GC and GC-MS analysis. GC and GC-MS-Fatty acid methyl esters were analyzed in a Perkin-Elmer 990 gas chromatograph using a 10% EGSS-X column (100-200 mesh, Applied Sciences Laboratories). An isothermal program at 150 "C was employed with helium as carrier gas. The methyl esters were identified by their retention times relative to the methyl n-eicosanoic acid (C20) internal standard which was used both for quantitation and mass spectral analysis.
The amount of fatty acid present per mol of protein was based on the values obtained from these analyses and from the quantity obtained from amino acid mol/ mol calculation.
The GC-MS analysis was done with Hewlett-Packard Quadruple mass spectrometers (models 5982A and 5980A) coupled to ion selector devices. The instrument was operated with an electron energy of 40 eV in the ionization mode using a 5-ft glass column (0.4 cm inner diameter) packed with 3% OV-1 (Supelco) on 80/100 Supelcoport.
A temperature program of 150-280 "C at 6.0 degrees/min was employed.
Helium was used as carrier gas at a flow rate of 25 ml/ min. The ion source and jet separator were both at 260 "C.

RESULTS
Purification and Amino Acid Composition-Ligatin purified by gel filtration is a homogeneous species of M, = 10,000 as shown by SDS-PAGE ( Fig. 1) (2, 3). When subjected to rpHPLC, this protein was eluted as a single peak at 39% CH,CN under neutral conditions (Fig. 2). No other material absorbing at 214 nm was recovered. The recovery of ligatin in multiple runs of the same preparation from this column was found to vary from 40 to 80%. The lower yields were obtained with more concentrated samples and after prolonged storage of the protein at -70 "C. This purified polypeptide was 5 blocked to Edman degradation. The amino acid composition of ligatin isolated by standard purification techniques and by reverse-phase chromatography is compared in Table I. The compositions are similar in that each contains predominantly aspartic acid, glutamic acid, glycine, and serine (see Table I) but differ in the amounts of isoleucine, leucine, and phenylalanine.
This increase in the amount of hydrophobic residues was consistently observed in samples subjected to reverse-phase chromatography. Acidic and hydroxyamino amino acids account for almost 50% of the total residues and no tryptophan was detected by uv spectral analysis. Calculation based on the number of residues of each amino acid suggests a polypeptide of 100 residues with a molecular weight of 10,010. This is consistent with our previous estimate of M, = 10,000 by electrophoretic analysis (2, 3). Ligatin preparations also contain ninhydrin positive material which was eluted as three peaks that do not correspond  *Although methionine was detected, the protein was not sensitive to cyanogen bromide cleavage and performic acid oxidation did not destroy this compound. e ND, not detected.
to the common amino acids, ammonia, or reagent background. Two of these peaks were eluted before threonine indicating that they are highly acidic substances. The third component is relatively basic and appears just before lysine. One of the two acidic peaks corresponds to the elution position of phosphoserine (2). The other acidic peak was eluted between aspartic acid and threonine. The identity of this acidic peak and the basic peak are currently unknown. Fatty Acid Acylation: GC-MS-Ligatin's solubility in acidified ch1oroform:methanol and its ability to interact directly with a CI resin suggested a hydrophobic character that cannot be readily explained by its amino acid composition. Since other eukaryotic membrane-bound proteins are known to contain covalently bound fatty acids, we investigated ligatin preparations for this chemical modification. Two types of ligatin preparations were analyzed for fatty acylation: one that had been dialyzed extensively against HnO and a second that had been extracted with organic solvents and subjected to affinity chromatography on a neomycin-glass support. The polypeptide-associated fatty acids from each preparation were subsequently removed by transesterification and analyzed by GC and GC-MS. Fig. 3 shows a typical ion tracing of the ligatin preparations. Both preparations showed the presence of methyl palmitate (Fig. 34. The spectra and retention times obtained were identical to those of published standards. The amount of methyl palmitate was quantitated (Table 11) and found to be 1-2 mo1/10,000 g of protein. A minor amount (0.02-0.4 mol/mol of polypeptide) of methyl oleate was also detected (Fig. 3b).
Interaction with Lipid Bilayers-To investigate the interaction of ligatin with membrane lipids, we tested its ability to alter the conductance of bimolecular lipid films in uitro. Two types of bilayers were constructed phosphatidylcholine

FIG.
3. Mass spectra of ligatin-associated fatty acids removed by transesterification. The fatty acids were isolated and esterified as described under "Experimental Procedures." The methyl esters were separated using a column packed with 3% OV-1 on 80/ 100 Supelcoports and analyzed with a Hewlett-Packard mass spectrometer. The spectra ( a and b) and retention times obtained were identical to that of standard methyl palmitate and methyl oleate, respectively. A.M.U., atomic mass units.

TABLE I1
Fatty acids associated with rat ileal ligatin Fatty acids were removed by methanolic alkali and analyzed as methyl esters by gas chromatography. Given is the amount of fatty acid present in two separate preparations quantitated relative to neicosanoic acid (C20) internal standard. ( Fig. 4). Ligatin at 8 Fg/ml increased the membrane conductance by 3-fold. In contrast, when phosphatidylserine was used as the membrane-forming lipid, no change in membrane conductance occurred upon the addition of ligatin. The experiment with deacylated ligatin was not done because even under mild deacylation conditions the amino acid analysis indicated the peptide had been altered.

DISCUSSION
In the studies reported here, we show that ligatin is an acyl protein, composed of a polypeptide of M, = 10,000 which is a single peak in reverse-phase high performance liquid chromatography (rpHPLC). Amino acid analysis shows that ligatin has a high content of polar residues, suggesting that its amphipathic properties are not due to its protein component. Two types of bilayers were tested: phosphatidylcholine (C160 34%; C181 35%) (0) and phosphatidylserine (C180 42%; C181 37%) (0). All aqueous solutions were unbuffered 0.1 M NaCl. Ligatin was present in both chambers.
as palmitic acid and the content as 1.4-1.7 mo1/10,000 g of protein.
Fatty acids are released from purified ligatin under mild methanolic alkaline conditions, suggesting that they are covalently bound through ester linkages. The composition of the protein-bound fatty acids seems to account for ligatin's selective location at the external leaflet of the membrane. Palmitic acid is bound to the protein in a 1-2 mo1:mol ratio; oleic acid which is consistently observed is bound to the polypeptide in a submolar concentration (0.02-0.4 mol:mol polypeptide). Other acyl proteins are known to contain palmitic acid (11)(12)(13)(14)(15)(16) covalently attached in an amino acid-ester linkage (11,(13)(14)(15). The potential acylation sites are found close to the postulated membrane-spanning segments of these int.egra1 membrane proteins (15)(16)(17). In contrast, ligatin's extraction in the absence of detergents suggests it lacks a transmembrane domain. These findings suggest the existence of a novel site, the E X 0 domain of a membrane protein for fatty acylation.
The covalent attachment of lipid to cell surface proteins which contain only an E X 0 domain has been described (18)(19)(20). This post-translational modification, consisting of phosphatidylinositol containing glycolipid covalently linked to the carboxyl terminus, functions as a membrane anchor (18,20). The two fatty acids attached to phosphatidylinositol have been identified as myristic acid (18,19). Since this posttranslational modification appears on a subclass of glycoproteins that have hydrophobic properties yet do not contain any extended sequence of hydrophobic amino acids (18,19), we are exploring the possibility that phosphatidylinositol containing glycolipid is present on ligatin and may be the site of fatty acylation.
Alternately fatty acids may be esterified to serine and threonine residues of a polypeptide chain and provide an anchorage site within the lipid bilayer (16,(21)(22)(23). In support of this hypothesis, we have found that ligatin increases the conductance across neutral phosphatidylcholine bilayers in a concentration-dependent manner. Such a change is dependent on a perturbation of the fatty acid region of the bilayer, whereas an interaction with the head group region alone does not affect the conductance (24). These results lend validity to the notion that in vitro ligatin most likely interacts directly with the lipid core and not to the head group region of the membrane phospholipids. Since ligatin has a low content of hydrophobic amino acids, the fatty acids covalently bound to ligatin may be responsible for ligatin's attachment and stabilization in membranes.
The covalent attachment of palmitic acid to ligatin distinguishes it from Ca2+-binding proteins such as calmodulin ( 2 5 ) , troponin C (25), calcineurin B (21,25), and SlOO (26). These proteins are members of a group of homologous Ca'+-binding proteins that function as secondary messengers in regulating metabolic and contractile activities within a cell (25,27). They are found in varying concentrations in all vertebrate tissues (27) and are distributed between the soluble and particulate fractions dependent upon the Ca'+ concentration present during isolation (27). The Ca'+-binding proteins are released from membranes by EGTA at pH 8. In contrast, ligatin separates from the membrane in the presence of Ca'+ (5 mM or greater) at neutral pH values but can be released by pH 8.0 alone. Furthermore, ligatin differs from the Ca"binding proteins in its cellular location at the external leaflet of the plasma membrane (1-3) and its physiological function as a baseplate for the attachment of a subclass of phosphoglycoproteins at the cell surface (1, 3, 4). These studies now provide additional criteria for the identification of ligatin based on its characterization as an acyl protein. Fatty acids have not been detected in Ca2+-binding proteins with the exception of calcineurin B which contains myristyl groups (28).
A second group of membrane proteins with which ligatin has been confused because of similarities in putative physiological function as a baseplate for phosphoglycoproteins is the family of glucose 1-phosphate-binding proteins (29,30). Again, ligatin can be distinguished by its size, its acylation, and its solubility in acidified organic solvents. Most notable, the glucose 1-phosphate-binding proteins are visualized in SDS-polyacrylamide gels by Coomassie Blue following standard fixation protocols. In contrast, ligatin is soluble in acidified methanol used to precipitate polypeptides within polyacrylamide gels (31) and therefore, it cannot be visualized by this protocol.
Clearly, ligatin meets the criteria for a peripheral membrane protein because of its polar amino acid composition and also because of its release from membranes by cations and pH (4.5 and 8.0) (1) which perturb ionic interactions. Yet, ligatin inserts into black lipid films in vitro in a noncovalent hydrophobic manner characteristic of integral membrane proteins.
Therefore, ligatin represents a subclass of periphera1 membrane proteins, one that exhibits amphipathic properties critical for protein-lipid interactions but one that partitions into either a hydrophobic or a hydrophilic domain dependent upon pH or cations. It is postulated that pH-induced changes enable this protein either to interact with target membranes or to separate from the membrane, thereby providing a mechanism for regulating ligatin's expression at the membrane surface.