Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family

We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine alpha-amide, and four cysteine residues forming two disulfide bridges. This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions. Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes. Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.     

Molecular cloning, expression analysis and cellular localization of gomesin, an anti-microbial peptide from hemocytes of the spider Acanthoscurria gomesiana

Gomesin is a cationic anti-microbial peptide of 18 amino acid residues isolated from the hemocytes of unchallenged tarantula spider Acanthoscurria gomesiana. This paper reports the first study of the processing and cellular location of an anti-microbial peptide (AMP) in spiders. Gomesin cDNA sequence analysis indicated that it is processed from a precursor containing a signal peptide (23 amino acid residues) and a negative C-terminal region (43 amino acid residues). The gomesin gene was constitutively transcribed in hemocytes and the gene product localized in hemocyte granules. The constitutive production of gomesin by a spider is discussed in the context of an ancient mechanism of AMP regulation and storage.     

Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability

Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.     

Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity

The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.     

A cyclic peptide inhibitor of apoC-II peptide fibril formation: mechanistic insight from NMR and molecular dynamics analysis

The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation.     

NMR structures of the C-terminal segment of surfactant protein B in detergent micelles and hexafluoro-2-propanol

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.     

Gomesin, a peptide produced by the spider Acanthoscurria gomesiana, is a potent anticryptococcal agent that acts in synergism with fluconazole

Gomesin is an 18-residue cysteine-rich antimicrobial peptide produced by hemocytes of the spider Acanthoscurria gomesiana. In the present study, the antifungal properties of gomesin against Cryptococcus neoformans, the etiologic agent of cryptococcosis, were evaluated. Gomesin bound to the cell surface of cryptococci, which resulted in cell death associated with membrane permeabilization. Antifungal concentrations of gomesin were not toxic for human brain cells. Supplementation of cryptococcal cultures with the peptide (1 microM) caused a decrease in capsule expression and rendered fungal cells more susceptible to killing by human brain phagocytes. The possible use of gomesin in combination with fluconazole, a standard antifungal drug, was also evaluated. In association with fluconazole, gomesin concentrations with low antimicrobial activity (0.1-1 microM) inhibited fungal growth and enhanced the antimicrobial activity of brain phagocytes. These results reveal the potential of gomesin to promote inhibition of cryptococcal growth directly or by enhancing the effectiveness of host defenses.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Effect of the antimicrobial peptide gomesin against different life stages of Plasmodium spp

While seeking strategies for interfering with Plasmodium development in vertebrate/invertebrate hosts, we tested the activity of gomesin, an antimicrobial peptide isolated from the hemocytes of the spider Acanthoscurria gomesiana. Gomesin was tested against asexual, sexual and pre-sporogonic forms of Plasmodium falciparum and Plasmodium berghei parasites. The peptide inhibited the in vitro growth of intraerythrocytic forms of P. falciparum.When gomesin was added to in vitro culture of P. berghei mature gametocytes, it significantly inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anopheles stephensi mosquitoes, and did not appear to affect the mosquitoes. These properties make gomesin an excellent candidate as a transmission blocking agent for the genetic engineering of mosquitoes.     

Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.     

Recombinant Fusion Proteins for the Industrial Production of Disulfide Bridge Containing Peptides: Purification, Oxidation without Concatamer Formation, and Selective Cleavage

We report the biotechnical production of peptides of approximately 35–50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach. This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation. The fusion tail described here serves several purposes: (i) it enables high expression levels inEscherichia colito be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide. A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide. Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide. The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides.     

Evidence for a Partially Structured State of the Amylin Monomer

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.     

Charge delocalisation and the design of novel mastoparan analogues: enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP

The formation of an amphipathic helix is a major determinant of the biological activity of the tetradecapeptide mastoparan (MP). To address the functional significance of lysyl residues at positions 4, 11 and 12 of MP, we synthesised five novel analogues using sequence permutation and arginine-substitution to delocalise cationic charge. Comparative bioassays determined cytotoxicity, beta-hexoseaminidase secretory efficacy and peptide-activated extracellular receptor-stimulated kinase (ERK)1/2 phosphorylation. The monosubstitution of individual lysine residues with arginine produced differential changes to the indices of cytotoxicity and secretion indicating that these conservative substitutions are compatible with membrane translocation and the selective binding and activation of intracellular proteins. More profound changes to the predicted hydrophilic face of MP, resulting from the relocation or substitution of additional lysyl residues, enhanced both the cytotoxicity and secretory efficacy of novel peptides. Significantly, the more amphipathic peptide [Lys5, Lys8, Aib10]MP was identified to be both the most cytotoxic and the most potent secretagogue of all the peptides compared here. Charge delocalisation within the hydrophilic face of MP analogues was also compatible with peptide-induced activation of ERK1/2 phosphorylation. Our data indicate that charge delocalisation is a suitable strategy to engineer more potent analogues of MP that differentially target intracellular proteins.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.     

Isolation and purification of the extracellular and intracellular portions of the beta subunit of (Na+,K+)-ATPase.

The beta subunit of lamb kidney (Na+,K+)-ATPase was isolated by size exclusion high performance liquid chromatography. Treatment of the beta subunit with formic acid yielded two peptide fragments which were purified via reversed phase high performance liquid chromatography. These peptides were identified by sodium dodecylsulfate polyacrylamide gel electrophoresis, amino acid analysis and N-terminal sequencing as (Pro 94-Ser 302), a largely hydrophilic peptide which comprises the major portion of the extracellular domain including six Cys residues which participate in disulfide bond formation and three glycosylation sites and a smaller peptide (Ala 1-Asp 93) which contains the single membrane spanning region and the intracellular domain.