Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active beta subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.     

Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes

The 20 S proteasome processively degrades cell proteins to peptides. Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation. Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length. The average size of peptides generated from these four polypeptides was 8 +/- 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods. However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a log-normal distribution. No length was predominant, and more than half were shorter than 10 residues. Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues. These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids.     

Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.     

Contribution of proline residue for efficient production of MHC class I ligands by proteasomes

Proteasomes are processing enzymes capable of generating major histocompatibility complex (MHC) class I ligands, but the mechanism of how they excise ligands without destroying them is largely unknown. Previously, we reported that most products of ornithine decarboxylase degraded in vitro by the 26 S ATP-dependent proteasome, which contained one or two Pro residues (Tokunaga, F., Goto, T., Koide, T., Murakami, Y., Hayashi, S., Tamura, T., Tanaka, K., and Ichihara, A. (1994) J. Biol. Chem. 269,17382-17385), which implied that the Pro residue has a role in the escape from random cleavage by proteasomes. Here, we examine the role of the Pro residue in producing MHC class I ligands in vitro. Proteasomes generated two cytotoxic T lymphocyte-epitopic precursor peptides, SIIPGLPLSL and DMYPHFMPTNL, from the 29-mer and 25-mer peptides harboring these sequences, which are derived from the c-akt proto-oncogene and the pp89 protein of mouse cytomagalovirus, respectively. Replacement of the first or second Pro residue within these epitopes by Ala resulted in a marked reduction of this epitope-derived production or their random cleavage by proteasomes, irrespective of the presence of PA28, which greatly accelerates the generation of unmodified ligands. Moreover, replacement of a single amino acid residue other than Pro in both epitopic and flanking regions by Ala or Leu had no or little appreciable effect on the SIIPGLPLSL or its derivative production. Thus, Pro residue(s) within these epitopic sequences presumably contributes to efficient production of MHC class I ligands through prevention of their random cleavage by proteasomes.     

Proteasomes generate in vitro a natural peptide of influenza-A nucleoprotein functional in HLA-B27 antigen assembly

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.     

Molecular mechanisms of class I major histocompatibility complex antigen processing and presentation

The presentation of antigenic peptides by class I major histocompatibility complex molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by CD8+ T lymphocytes which inspect peptides, derived from intracellular proteins, bind to class I molecules on the surface of most cells. The transporter associated with antigen processing selectively translocates cytoplasmically derived peptides of appropriate sequence and length into the lumen of the endoplasmic reticulum where they associate with newly synthesized class I molecules. The translocated peptides are generated by multicatalytic and multisubunit proteasomes which degrade cytoplasmic proteins in a ATP-ubiquitin-dependent manner. This review discusses our current molecular understanding of class I antigen processing and presentation.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation.     

Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana)

The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.     

The amphipathic helix concept: length effects on ideally amphipathic LiKj(i=2j) peptides to acquire optimal hemolytic activity

In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.     

Ovine submaxillary mucin. Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase

Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase.     

Horsetail (Equisetum telmateia) ferredoxins I and II. Amino acid sequences

Two ferredoxins were isolated from horsetail (Equisetum telmateia) and their amino acid sequences were determined by use of a sequence analyzer in combination with carboxypeptidase digestion and manual Edman degradation of tryptic peptides of carboxymethyl-ferredoxins. Ferredoxins I and II each had only four cysteine residues in a total of 95 and 93 residues, respectively. The amino-terminal residues of both ferredoxins were heterogeneous, but alanine was concluded to be their genuine terminal residue. The comparison of these isozymelike molecules showed 29 differences in amino acid residues with three inverted replacements. One gap was inserted in ferredoxin II at position 32 to align the ferredoxins with greatest homology. Despite the many differences in amino acid residues there was no difference in net charges of the two ferredoxins.     

Structure and action of heteronemertine polypeptide toxins. Primary structure of Cerebratulus lacteus toxin B-IV

The amino acid sequence of Cerebratulus toxin B-IV, a crustacean-selective axonal toxin occurring in the marine worm C. lacteus, was determined by Edman degradation of the tryptic and staphylococcal protease peptides obtained from the reduced, carboxymethylated toxin. All four of the anticipated maleylated tryptic peptides, ranging in size from 8 to 23 residues, and three staphylococcal protease peptides, ranging from 9 to 35 residues, were isolated in pure form by gel filtration followed by either ion exchange chromatography or preparative paper electrophoresis. The order of the maleylated tryptic peptides was based upon the sequences of the staphylococcal protease peptides. As might be expected, toxin B-IV displays no homology with the elapid nicotinic receptor toxins. In addition, toxin B-IV is structurally unrelated to a group of scorpion neurotoxins which, like B-IV, affect action potential generating mechanisms.     

The making of the dominant MHC class I ligand SYFPEITHI

The proteasome contributes to the generation of most of the peptide ligands of MHC class I molecules. To compare the identity of the peptides generated by the proteasome with those finally presented by MHC class I molecules, we generated a monoclonal antibody recognizing the C-terminal part of the dominant H2-Kd ligand SYFPEITHI derived from the JAK1 tyrosine kinase. Immunoprecipitations of lysates from H2-Kd-expressing or non-expressing cells revealed that only in the presence of H2-Kd SYFPEITHI could be isolated. No longer potential precursor peptide containing SYFPEITHI could be detected. Surprisingly, a peptide lacking the first two amino acids, FPEITHI, was isolated independently of the presence of H2-Kd molecules. The detection of only SYFPEITHI and FPEITHI in cell lysates corresponded with the strong generation of these two peptides in in vitro digests of elongated SYFPEITHI-containing peptides with purified 20S proteasomes. Our results indicate that MHC ligands can be generated directly by the proteasome in vivo and that at least for SYFPEITHI the expression of the corresponding MHC molecule is critical for protection of the ligand in vivo.     

Studies on the variability of growth and proportions of the metacarpals at postpubertal age

Bovine lutropin (luteinizing hormone) was carboxymethylated at pH3.0 for 12 h at 37 degrees C with iodoacetic acid for specific modification of methionine residues. To facilitate the location of preferentially modified methionine residues, iodoE114C]acetic acid was added as tracer. The alpha and beta subunits of bovine lutropin were carboxymethylated with a 2- or 5-fold molar excess of iodoacetic acid either in the presence or absence of their counterpart subunits. The modified subunits were separated and isolated by counter-current distribution followed by gel filtration on Sephadex G-100. To locate the modified methiones, the isolated alpha or beta chain was reduced. S-carboxymethylated and subjected to tryptic hydrolysis. The tryptic peptides were fractionated by gel filtration on Bio-Gel P-10. From analyses of the purified 14C-labelled tryptic peptides, it was observed that methionine-8 and -33 in bovine lutropin alpha chain and methionine-52 in the beta chain were preferentially modified. Similar results were obtained when isolated alpha and beta subunits were individually carboxymethylated in the absence of their counterpart subunit under identical conditions. The fact that a recombinant of native human lutropin alpha chain, in which a valine residue is present in the position corresponding to methionine-8 of bovine lutropin alpha chain, and carboxymethylated bovine lutropin beta chain regenerated a substantial amount of receptor-site-binding activity indicated that methionine-8 in bovine alpha chain was biologically not essential. These studies showed clearly that both methionine-33 in the alpha chain and methionine-52 in the beta subunit were involved for optimum binding between bovine lutropin and its receptors for expression of hormonal activity.     

A molecular basis for how a single TCR interfaces multiple ligands

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.     

Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties

Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.     

The critical role of a solvent-exposed residue of an MHC class I-restricted peptide in MHC-peptide binding

The immunodominant ovalbumin257-264 (OVA-8, SIINFEKL) and herpes simplex virus gB496-503 (HSV-8, SSIEFARL) peptides share 50% amino acid identity (residues P1, P3, P5 and P8) and bind with comparable efficacy to the murine MHC-encoded class I molecule H-2Kb. However, these two peptides bind differently to H-2Kbm8, a natural H-2Kb variant with a substitution in four amino acids on the floor of the peptide-binding site; HSV-8 binds with high and OVA-8 with a relatively low efficacy. To investigate which of the non-homologous peptide residues were responsible for this differential binding, we used substituted peptide variants and the class I thermodynamic stabilization assay. Variation at the solvent-exposed peptide residues P6 and P7 did not appreciably influence binding. By contrast, variation at the buried P2 and, surprisingly, at the solvent-exposed P4 residue was found to be important. Transplantation of the HSV-8 P2 or P4 residues onto the OVA-8 backbone created variant peptides O2S (P2I-->S) and O4E (P4N-->E) that bound considerably better to H-2Kbm8 than OVA-8. Furthermore, the double-substituted peptide, O2S4E, bound even better, revealing a cooperative effect of the two residues. The reciprocally substituted peptides H2I and H4N, generated by grafting the OVA-8 P2 and P4 residues onto the HSV-8 backbone respectively, bound to H-2Kbm8 slightly worse than HSV-8 but the double-substituted peptide H2I4N bound as poorly as OVA-8. Effects exerted by the P4 residue, which is solvent accessible and therefore available for the TCR contact, demonstrated that exposed peptide residues can, in certain situations, influence not only the TCR contact but also MHC-peptide binding.     

Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching

A new method for the characterization of serine and threonine phosphorylation sites in proteins has been developed. After modification of a phosphoprotein by beta-elimination/ethanethiol addition and conversion of phosphoserine and phosphothreonine residues to S-ethylcysteinyl or beta-methyl-S-ethylcysteinyl residues, the modified protein was subjected to proteolytic digestion. Resulting digests were analyzed by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry and database searching to identify original phosphorylated residues. The computer program utilized (SEQUEST) is capable of identifying peptides and modified residues from uninterpreted MS/MS spectra, and using this method, all of the five known phosphorylation sites in bovine beta-casein were identified. Application of the method to multiply phosphorylated human high molecular weight neurofilament protein (NF-H) resulted in the identification of 21 peptides and their modified residues and hence, the in vivo phosphorylation sites. These included 26 KSP and 1 KTP site, all of which occur in the KSP repeat C-terminal tail domain (residues 502-823). One site at residue 518 was previously uncharacterized. A novel non-KSP serine at residue 421 near the KLLEGEE region in a IPFSLPE motif was characterized as phosphorylated (or glycosylated). The 27 characterized phosphorylation sites occur at S/TP residues in the following motifs: KSPVKEE, KSPAEAK, KSPEKEE, KSPAEVK, KSPEKAK, KSPPEAK, KSPVKAE, and KTPAKEE. On the basis of kinase consensus sequences, all of these motifs, including the previously unreported KTPAKEE motif, can be phosphorylated by proline-directed kinases. Advantages of the new method vis-a-vis our previously reported method [Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931-3940] include (i) production of diastereomers eluting at different retention times increased the chances of peptide identification, (ii) increased hydrophobicity and hence retention time of the modified peptides, (iii) facilitation of positive ion production, and (iv) increased susceptibility to tryptic digestion as a result of conversion of negatively charged phosphorylated residues to neutral S-ethylcysteine or beta-methyl-S-ethylcysteine residues.     

Haemoglobins of the shark, Heterodontus portusjacksoni II. Amino acid sequence of the alpha-chain

The amino acid sequence of the alpha-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble "core" peptide from the tryptic digestion contained 34 residues and required cleavage by several prosteases before the sequence was established. Compared with human alpha-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin. In the alpha1beta1 contact sites there are four changes in the oxyhaemoglobin form and six deoxy form. Nine of the 16, alpha1beta1 contact sites show variation while three of the haem contact sites have changed in comparison to the residues known to be involved in these interactions in horse haemoglobin alpha-chain. Use of the sequence data to estimate a time of divergence of the shark from the main vertebrate line yielded the value of 410 +/- 46 million years. The data, in general, support the palaeontological view that bony fishes arose before the elasmobranchs.