Expression,purification, crystallization,and preliminary X-ray analysis of recombinant human saposin B

Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes. Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable. We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies. The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen-deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A. Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2A. Despite obtaining only merohedrally twinned P3(1) native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3(1)21 was also grown. The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.     

Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).

Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.     

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.     

The cerebroside sulfate activator from pig kidney: Derivitization,cerebroside sulfate binding,and metabolic correction

Highly purified cerebroside sulfate activator from pig kidneys was characterized by a number of chemical and biological procedures. Methods for chemical modifications were evaluated in an attempt to obtain biologically active derivatives. Iodination, dabsylation, and to a lesser degree reductive methylation provided useful products with good retention of cerebroside sulfate activator activity. Other procedures resulted in largely inactive derivatives or losses in both protein and biological activities. Attempts at renaturation of cerebroside sulfate activator subjected to various denaturing conditions appeared to be successful in many instances, but it was uncertain if the protein structure had actually been disrupted. The binding of cerebroside sulfate by activator was estimated by gel filtration under conditions similar to those of its assay. The formation of a relatively stable 1:1 complex was observed, collaborating results with the human protein. The complex was stable enough to be isolated and shown to be an efficient substrate for arylsulfatase A. The effectiveness of the pig kidney cerebroside sulfate activator for correcting the metabolic defect in activator-deficient human fibroblasts was compared with human materials. The pig kidney protein was taken up more efficiently by the cells and resulted in a better metabolic correction than material from human liver, but was somewhat less effective than a preparation from human urine.     

Interaction of myelin basic protein and polylysine with synthetic species of cerebroside sulfate

The effect of myelin basic protein on the myelin lipid cerebroside sulfate was studied by differential scanning calorimetry and use of the fatty acid spin label, 16-S-SL, in order to determine (i) the effect of basic protein on the metastable phase behavior experienced by this lipid, and (ii) to determine if basic protein perturbs the lipid packing as it does with some acidic phospholipids. The effects of basic protein on the thermodynamic parameters of the lipid phase transition were compared with those of polylysine which has an ordering effect on acidic phospholipids as a result of its electrostatic interactions with the lipid head groups. Different synthetic species of cerebroside sulfate of varying fatty acid chain length and with and without a hydroxy fatty acid were used. The non-hydroxy fatty acid forms of cerebroside sulfate undergo a transition from a metastable to a more ordered stable state while the hydroxy fatty acid forms remain in the metastable state at the cation concentration used in this study (0.01 M Na+ or K+). The non-hydroxy fatty acid forms were still able to go into a stable state in the presence of both basic protein and polylysine. At low concentrations, basic protein increased the rate of the transition to the stable state, while polylysine decreased it for the longest chain length form studied. However, at high concentrations, basic protein probably prevented formation of the stable state. The hydroxy fatty acid forms did not go into the stable state in the presence of basic protein and polylysine. It is argued that the increased rate of formation of the stable state in the presence of basic protein and decreased rate in the presence of polylysine are consistent with interdigitation of the lipid acyl chains in the stable state. Basic protein also had a small perturbing effect on the lipid. It decreased the total enthalpy of the lipid phase transition. When added to the non-hydroxy fatty acid forms it increased the temperature of the liquid crystalline to metastable phase transition and decreased the temperature of the stable to liquid crystalline phase transition. It significantly decreased the transition temperature of the hydroxy fatty acid forms but only a portion of the lipid was affected. In contrast, polylysine increased the transition temperature of the metastable and stable states of all forms of cerebroside sulfate but had a greater effect on the non-hydroxy fatty acids forms than on the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. Deficiency in urine of patients affected with cerebroside sulfatase activator deficiency and identity with activators for the enzymatic hydrolysis of GM1 ganglioside and globotriaosylceramide

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.     

Microstructural polymorphism in bovine brain galactocerebroside and its two major subfractions.

Aqueous suspensions of either brain galactocerebrosides or its subfraction consisting of alpha-hydroxyacyl galactocerebrosides are mainly composed of vesicles or granular lipid with occasional multilamellar sheets. In aqueous media the other subfraction consisting of non-hydroxyacyl galactocerebrosides forms some helical structures, but most of the lipid remains as granules or vesicles. It is demonstrated that thermal cycling of non-hydroxyacyl galactocerebrosides in polar nonaqueous solvents can greatly enhance the degree of conversion to helical ribbons about 100 nm in diameter. These structures appear to be a stable dehydrated crystalline form of this lipid and are morphologically similar to helical microstructures produced by a few synthetic lipids. On the other hand, similar treatment of unfractionated bovine brain cerebroside and its alpha-hydroxy fatty acyl subfraction quantitatively produces straight needles that appear to be cochleate cylinders. While their dimensions depend on formation conditions, a typical suspension has uniform particles with diameters close to 100 nm and lengths variable from one to a few hundred micrometers. This is the first report demonstrating the quantitative formation of crystalline high axial ratio microstructures from complex mixtures of natural lipids. The different microstructures formed by the two components appear related to the various forms of lipid deposits occurring in lipid storage diseases. The similarity of these "synthetic" microstructures to biological structures in which they are found (such as myelin and intestinal brush border microvilli) strengthens the possibility that galactocerebrosides have a role in stabilizing cylindrical biological structures.     

Advances in membrane mimetics and mass spectrometry for understanding membrane structure and function

Membrane proteins and lipids play roles in regulating biological functions of cells. However, the analysis of interactions between membrane proteins and lipids in biological membranes remains challenging. Native membranes typically contain heterogenous lipid mixtures and low amounts of membrane proteins. This review presents recent developments in membrane mimetics and complementary mass spectrometry approaches for the investigation of membrane protein–lipid interactions after protein expression and purification. Furthermore, it is exemplified how delipidation knowledge on membrane mimetics can be used to gain insights into the role of lipids for protein structure and function. Because every technology has its strengths and weaknesses, it becomes apparent that integrated research approaches will facilitate the investigation of complex membrane environments in the future.     

Cyclotide-membrane interactions: defining factors of membrane binding, depletion and disruption

The cyclotide family of plant-derived peptides is defined by a cyclic backbone and three disulfide bonds locked into a cyclic cystine knot. They display a diverse range of biological activities, many of which have been linked to an ability to target biological membranes. In the current work, we show that membrane binding and disrupting properties of prototypic cyclotides are dependent on lipid composition, using neutral (zwitterionic) membranes with or without cholesterol and/or anionic lipids. Cycloviolacin O2 (cyO2) caused potent membrane disruption, and showed selectivity towards anionic membranes, whereas kalata B1 and kalata B2 cyclotides were significantly less lytic towards all tested model membranes. To investigate the role of the charged amino acids of cyO2 in the membrane selectivity, these were neutralized using chemical modifications. In contrast to previous studies on the cytotoxic and antimicrobial effects of these derivatives, the Glu6 methyl ester of cyO2 was more potent than the native peptide. However, using membranes of Escherichia coli lipids gave the opposite result: the activity of the native peptide increased 50-fold. By using a combination of ellipsometry and LC-MS, we demonstrated that this unusual membrane specificity is due to native cyO2 extracting preferentially phosphatidylethanolamine-lipids from the membrane, i.e., PE-C16:0/cyC17:0 and PE-C16:0/C18:1.     

The activator of cerebroside sulphatase. Binding studies with enzyme and substrate demonstrating the detergent function of the activator protein.

1. Sulphatase A (cerebroside sulphatase) (EC 3.1.6.1.) and a 12-fold excess of its physiological activator protein were chromatographed together on Sephadex G-75. The elution buffer was the same as that used in the enzymic degradation of sulphatides. The two proteins were eluted in different peaks indicating that no stable complex formed. 2. Activator protein was incubated with sulphatides under conditions used favouring the sulphatase activity. Incubation solutions were then examined by electrophoresis on a polyacrylamide gel gradient. An one-to-one complex between activator and sulphatides was observed. Half maximal binding occurred with 2.5 nmol of sulphatides together with 1 or 2 nmol of activator in 100 micronl. 3. Cerebrosides as the enzymic degradation products of sulphatides, bind also to the activator protein. A ratio of one-to-one could possibly be obtained at high cerebroside concentrations. The binding to cerebrosides is less specific than that to sulphatides. A 7-fold excess of cerebrosides was necessary for half maximal binding. 4. In a mixture of sulphatides and cerebrosides the formation of the complex with the activator protein is partly inhibited. The total amount of bound lipids changed as the composition of the lipid mixture was varied. In a one-to-one mixture of the two lipids 60% of the total bound lipids are sulphatides and 40% are cerebrosides.     

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well. An improved method of lipid extraction from chlorophyll-containing membranes is presented. This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction. The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography. The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography. The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis. None of the separated lipids alone was able to produce a well-coupled system. The best results were obtained with the native lipid mixture. The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol. Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included. Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C. The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.     

Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

Interaction of bradykinin with sodium dodecyl sulfate and certain acidic lipids

The striking change in the circular dichroism (CD) of bradykinin (BK) occasioned by its interaction with sodium dodecyl sulfate (SDS) is evidently due in large part to a change in the conformation of the C-terminal tetrapeptide moiety of the hormone. The full change in CD is induced by the binding of two molecules of monomeric SDS per peptide molecule, the complex being aggregated. Formation of the 1:2 BK-SDS complex apparently proceeds via intermediates of stoichiometry 1:1 and 2:1. The cooperative nature of the interaction is attributed to the SDS-promoted aggregation of BK. Electrostatic interactions with the Arg residues appear important for the binding reaction per se. CD reveals that BK also interacts with acidic lipids which bear a net electrical charge (e.g., cerebroside sulfate and phosphatidyl inositol) but not with lipids bearing no net charge (e.g., cerebroside and phosphatidyl choline). The interactions are with particular mixed micelles of the lipid and the nonionic surfactant used for their solubilization, micellar size and structure being examined by surface tensiometry and electron microscopy.     

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.     

Domain-induced activation of human phospholipase A2 type IIA: local versus global lipid composition

Secretory human phospholipase A2 type IIA (PLA2-IIA) catalyzes the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. The enzyme is coupled to the inflammatory response, and its specificity toward anionic membrane interfaces suggests a role as a bactericidal agent. PLA2-IIA may also target perturbed native cell membranes that expose anionic lipids to the extracellular face. However, anionic lipid contents in native cells appear lower than the threshold levels necessary for activation. By using phosphatidylcholine/phosphatidylglycerol model systems, we show that local enrichment of anionic lipids into fluid domains triggers PLA2-IIA activity. In addition, the compositional range of enzyme activity is shown to be related to the underlying lipid phase diagram. A comparison is done between PLA2-IIA and snake venom PLA2, which in contrast to PLA2-IIA hydrolyzes both anionic and zwitterionic membranes. In general, this work shows that PLA2-IIA activation can be accomplished through local enrichment of anionic lipids into domains, indicating a mechanism for PLA2-IIA to target perturbed native membranes with low global anionic lipid contents. The results also show that the underlying lipid phase diagram, which determines the lipid composition at a local level, can be used to predict PLA2-IIA activity.     

The cerebroside sulfate activator from pig kidney: purification and molecular structure.

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.     

Interaction between hepatic microsomal membrane lipids and apolipoprotein A-I

Incubation of apoprotein A-I (apo-A-I), the major protein component of human high density lipoprotein, with rat liver microsomal membranes under conditions of elevated pH and ionic strength leads to the production of a soluble protein:lipid complex (A-I/MM complex). The A-I/MM complex, as purified by density gradient centrifugation and agarose column chromatography, possesses a lipid composition similar to the hepatic microsomal membrane and a protein/lipid ratio similar to that of plasma high density lipoproteins, but markedly different from that of recombinant particles prepared with synthetic lipids. The A-I/MM complex constitutes a more physiological recombinant particle than can be formed using synthetic lipids and may be a suitable model for the newly assembled intracellular high density lipoproteins. Incubation of the erythrocyte plasma membranes with apo-A-I under the same conditions as used with microsomal membranes fails to generate any lipid:apoprotein complexes. This membrane specificity for forming soluble lipoprotein complexes suggests that the microsomal membranes possess a unique feature, possibly their lipid composition, which render them particularly suitable to serve as lipid donors to the apoproteins which are undergoing assembly within the endoplasmic reticulum/Golgi organelles.     

New sialic acid-containing sulfolipid: "ungulic acid"

Human epidermis, hair, nails, and kidney as well as bovine and horses' hooves were found to contain a lipid fraction, which on thin-layer chromatography migrated slightly ahead of the cerebroside sulfate esters and gave the color reaction specific for sialic acid. This fraction was isolated from horse hoof, in which it constituted nearly half of the total lipids. The purified fraction contained sulfur, but no phosphorus. The IR spectrum revealed the presence of a sulfate group, which was also determined by the benzidine method. Thin-layer and gas-liquid chromatography of the products of acid hydrolysis revealed the presence of sphingosine, galactose, galactosamine, and sialic acid. Fatty acid analysis showed that stearic acid was the major component, with minor amounts of palmitic and arachidic acids. The fraction isolated contained ceramide, sialic acid, galactose, galactosamine, and sulfate in equimolar amounts. We conclude that the new lipid is a ganglioside sulfate, which we have called "ungulic acid" because it was first separated and identified from a horse's hoof (Latin, ungula).     

Specific interaction of central nervous system myelin basic protein with lipids. Specific regions of the protein sequence protected from the proteolytic action of trypsin

The tryptic hydrolysis of the basic protein of central nervous system myelin (A₁ basic protein) and of A₁ basic-lipid complexes was studied. The tryptic digestion was monitored by “finger printing”, column chromatography and amino acid analysis of the resulting pure peptides.Specific regions of the protein sequence were found to be protected from the hydrolytic action of the trypsin only after the protein was recombined with specific lipids. The degree of protection was in the order: cerebroside sulphate > acidic lipid fraction of myelin > phosphatidylsrine = total lipid extract of myelin. The protected Lys-X, Arg-X bonds were all situated in the region amino acid 20 to amino acid 113 of the intact protein. This region contains the (proline)₃ bend in the protein which is stabilized by interaction with lipids and also the encephalitogenic site for monkey and rabbit.From the results reported in this publication we would like to suggest a specific interaction between a region of the A₁ basic protein molecule and cerebroside sulphate. Differences in A₁ basic protein-lipid interaction in different animals arising from differences in lipid composition and fatty acid composition of the different lipid species combined with minor changes in the protein sequence could explain the species variability of the encephalitogenic sites of the A₁ basic protein.