Neurotrophic actions of a novel molluscan epidermal growth factor.

The mammalian epidermal growth factor (EGF) is expressed in the developing and adult CNS, and it has been implicated in the control of cell proliferation, differentiation, and neurotrophic events. Despite extensive evolutionary conservation of the EGF motif in a range of different types of proteins, secreted EGF homologs with neurotrophic actions have not been reported in invertebrates. In this study, we present a novel member of the family of EGF-like growth factors, an EGF homolog from the mollusc Lymnaea stagnalis (L-EGF), and we demonstrate that this protein has neurotrophic activity. Purified L-EGF is a 43-residue peptide and retains the typical structural characteristics of the EGF motif. The L-EGF cDNA reveals a unique precursor organization. In contrast to the multidomain mammalian EGFs, it consists of only two domains, a signal peptide and a single EGF motif. Conspicuously, the L-EGF precursor lacks a transmembrane domain, setting it apart from all other members of the EGF-family. L-EGF mRNA is expressed throughout embryonic development, in the juvenile CNS, but not in the normal adult CNS. However, expression in the adult CNS is upregulated after injury, suggesting a role of L-EGF in repair functions. This notion is supported by the observation that L-EGF evokes neurite outgrowth in specific adult Lymnaea neurons in vitro, which could be inhibited by an EGF receptor tyrosine kinase inhibitor. In conclusion, our findings further substantiate the notion that the EGF family has an early phylogenetic origin, and our data support a neurotrophic role for L-EGF during development and injury repair.

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

Quantification of jasmonic acid, methyl jasmonate, and salicylic acid in plants by capillary liquid chromatography electrospray tandem mass spectrometry.

Jasmonic acid, methyl jasmonate, and salicylic acid have been reported to occur in plants and are thought to be essential for the regulation of systemic defense responses. This work describes a method for the quantitation in plant tissue of these regulators by reverse-phase capillary liquid chromatography interfaced to an electrospray tandem mass spectrometer. Inclusion during sample preparation of hydrogenated and/or deuterated internal standards corresponding to analogs of the regulators compensated for sample loss and permitted quantitation using the multiple reaction monitoring mode of the mass spectrometer. The free acids were analyzed in a negative-ion mode, whereas methyl jasmonate was analyzed in a positive-ion mode. Using these procedures an extract of fresh hybrid poplar leaves was found to contain per gram of leaf tissue 2.6 micrograms of jasmonic acid, 1.3 micrograms of methyl jasmonate, and 31.0 micrograms of salicylic acid. The techniques used should be applicable to other plant materials.

Stability enhancement for peptide analysis by electrospray using the triple quadrupole mass spectrometer.

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 microm i.d. by 145 microm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 microL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 microm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-microm column at a flow rate of 150 to 200 nL/min.

Primary structure of a new neuropeptide, cerebral peptide 2, purified from cerebral ganglia of Aplysia.

We report the purification and characterization of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed cerebral peptide 2 (CP2) because it was the larger of two peptides predominantly synthesized in the cerebral ganglia and transported to other regions of the central nervous system. The purification of CP2 from extracts of cerebral ganglia using three sequential modes of high-pressure liquid chromatography (HPLC) was followed using the [35S]methionine-labeled peptide obtained from transport experiments. The primary structure of CP2 was determined by automated Edman degradation of native CP2 and its proteolytic fragments in conjunction with mass spectrometry. CP2 is a 41 amino acid peptide with an amidated carboxyl terminal. A peptide with the proposed sequence of CP2 was synthetized and compared by HPLC with the native peptide. Chromatographic properties of the synthetic and native peptide labeled in vivo were found to be identical. CP2 does not appear to be a member of any previously identified peptide family.

Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry.

Low-energy negative-ion electrospray mass spectrometry (ESI-MS) and ESI-MS/MS were used to characterize saturated and unsaturated fatty acids. The carbon number and degree of unsaturation of fatty acids were determined using ESI-MS, and MS/MS was used to localize some double bond positions of mono-and polyunsaturated fatty acids. For compounds with up to two unsaturated bonds, fragmentation was dominated by loss of H2O from the carboxyl moiety and very low-intensity peaks generated from bonds cleaved at carbons alpha and/or beta to sites of unsaturation. Fragmentation of monounsaturated fatty acids was minimal using this soft method of mass spectrometric analysis, but increased with progressively greater degrees of fatty acid unsaturation. There was extensive hydride migration during ESI-MS/MS of compounds with three or more double bonds. Although this behavior complicated localization of double and triple bonds, the spectra were reproducible. Many peaks could not be definitively assigned to specific product ions, but the spectra of standards and complementary natural products were similar and isobaric compounds could be differentiated. The utility of this technique to examine biological samples was shown by analysis of the fatty acid composition of cod liver oil. Detection limits for negative-ion ESI-MS/MS were at or below 1 pg.

Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.

Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.

This paper describes the use of positive and negative ion electrospray mass spectrometry (MS) and MS/MS (tandem mass spectrometry) to identify glycerophospholipid and ceramide headgroups and their alkyl, alkenyl and acyl constituents. Molecular ion adducts were the primary products formed by positive ionization, occurring as [M+H]+, [M+Na]+, [M+K]+, [M+formate]+, or [M+acetate]+, depending upon the class of glycerophospholipid and the presence or absence of these ionization-promoting species. Similar (negatively charged) ions corresponding to the loss of the groups listed above were formed in negative ion MS. Positive ion electrospray MS/MS provides information on the nature of the headgroup, with the formation of an ion corresponding to the headgroup itself, or the loss of the headgroup from the molecular ion H+ or Na+ adduct. Acyl constituents are identified during negative ion MS/MS from the formation of their RCOO- ions. The nature of alkyl or alkenyl substituents in glycerophosphoethanolamine (PE) molecular species can be identified from residual ions following the loss of ethanolamine plus loss of the acyl moiety in the sn-2 position, and cyclization of a phosphate oxygen with C-2 of glycerol. In glycerophosphoinositol (PI) species, it appears that an RCO- ion is formed during negative ion MS/MS, possibly to steric interference from the bulky phosphoinositol headgroup that prevents cyclization (and subsequent stabilization) of the ion described for PE species. Positive and negative ion electrospray MS spectra for molecular species of commercial preparations of PE, PI, phosphatidylserine (PS), glycerophosphocholine (PC) and sphingomyelin (SM) produced similar profiles. For phospholipids occurring as Na+ adducts, concentrations above ca. 1 ng/microliter produced significant quantities of both [M+H]+ and [M+Na]+ ions for those molecular species present in the largest quantities, complicating interpretation of the spectra. Complete profiles of molecular species were obtained from as little as 10 picograms of material. Major components of PE were identified from 0.1 picogram total lipid. Using single ion monitoring of the Na+ adduct of beta-acetyl-gamma-O-hexadecyl L-alpha-phosphatidylcholine, 10 femtograms of material was detected. A mixture of 1 nanogram each of PE, PI, PS, and PC was readily resolved into individual molecular species, with little apparent loss of resolution or preferential ionization. Electrospray MS did not provide information on the position (sn-1 or sn-2) of fatty acids, and was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without prior separation of these lipid subclasses.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of rhodopsin multiple phosphorylation.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor at multiple sites near the C-terminus by rhodopsin kinase and binding of a regulatory protein, arrestin. In the present study, the phosphorylation sites were examined in a partially reconstituted system under several experimental conditions. Initial phosphorylation sites were found to be 338Ser, 343Ser, and 334Ser based on analysis by mass spectrometry of proteolytic peptides from the C-terminus. The extent of phosphorylation was found to be limited by two mechanisms: (1) binding of arrestin to phosphorylated rhodopsin (one to three phosphate groups) appeared to prevent further phosphorylation (arrestin has also been observed to promote the initial phosphorylation of rhodopsin at 338Ser in rod outer segment homogenates); and (2) reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol prevented phosphorylation at more than three sites. We propose that previous observations of higher levels of rhodopsin phosphorylation may be the result of the removal of endogenous arrestin, or of exceeding the capacity of retinol dehydrogenase activity by intense bleaches (e.g., by exhausting endogenous NADPH).

Sequential phosphorylation of rhodopsin at multiple sites.

Photolyzed rhodopsin is phosphorylated at multiple serine and threonine residues during the quenching of phototransduction. Sites of phosphorylation by rhodopsin kinase have been localized to the C-terminal region of rhodopsin, but no information was available on the kinetics and identity of phosphorylated residues. To determine the kinetics of phosphorylation at specific residues, the phosphorylated C-terminal peptide of rhodopsin (330DDEASTTVSKTETSQVAPA) obtained by proteolysis of rhodopsin with endoproteinase Asp-N was subjected to further subdigestion followed by electrospray mass spectrometry. Analysis of monophosphorylated peptide revealed that the major initial phosphorylation site is 338Ser. The analysis of di- and triphosphorylated peptides indicated that 343Ser or 336Thr residues are subsequent phosphorylation sites. These three residues, located in the C-terminal region of rhodopsin, are likely to be key phosphorylation sites of rhodopsin during the quenching of phototransduction. Identification of the kinetics of phosphorylation will facilitate understanding the functional significance of rhodopsin phosphorylation at multiple sites and the mechanism of rhodopsin kinase action.

The NH2 terminus of retinal recoverin is acylated by a small family of fatty acids.

Recoverin is a recently identified Ca(2+)-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.

Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2.

To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.

Collagen type IX: evidence for covalent linkages to type II collagen in cartilage.

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.

Primary structure of duck amyloid protein A. The form deposited in tissues may be identical to its serum precursor.

The amino acid sequence has been determined for the major protein that accumulates in amyloid fibrils in tissues of the Pekin duck. With the exception of 16 residues at the amino terminus, this 106-residue protein is homologous with human serum amyloid protein A (104-residue apoSAA), which is the putative precursor of the 76-residue protein that accumulates in human patients with amyloidosis. Duck serum is shown to contain a protein that is immunologically related and approximately equal in size (12 kDa) to the deposited form in ducks. These results indicate that proteolytic processing of the precursor is not a necessary step in the deposition of amyloid fibrils, at least in the duck.

Amino acid sequence of human von Willebrand factor.

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.

Molecular organization of the sex steroid-binding protein (SBP) of human plasma.

Several years ago this laboratory presented evidence that SBP is a dimer composed of two subunits having similar molecular weights. The question of whether or not these subunits are identical and therefore products of a single gene remained unanswered. We now report that the two polypeptide chains are identical and that SBP is a homodimer. The experimental approach was to reduce and [14C]alkylate cystine residues in human SBP, digest the product with trypsin or cyanogen bromide and determine the number of unique amino acid sequences around each [14C]carboxymethylcysteine residue. Only four unique sequences were found when all the radioactive peptides were analyzed. Since there are eight half-cystine residues per dimer, the results support a homodimeric structure.

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II.

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.

Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8.

The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented. The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides. The sequence is homologous to c-type cytochromes, especially in the heme-binding region.

Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase.

The complete amino acid sequence of the catalytic subunit (gamma subunit) of rabbit skeletal muscle phosphorylase b kinase was determined. The gamma subunit was purified by gel filtration in acidic 8 M urea after reduction and S-carboxymethylation in 7 M guanidine hydrochloride. Cleavage of the gamma subunit at arginyl bonds gave a complete set of nonoverlapping peptides. Overlapping peptides were obtained by cleavage at methionyl, tryptophanyl, or glutamyl bonds and by selected subdigestion of two large peptides obtained by cleavage at methionyl bonds. Sequence analysis established that the protein contains 386 residues corresponding to a molecular weight (Mr) of 44673. Comparison of the gamma subunit with the catalytic subunit of bovine cAMP-dependent protein kinase and with tyrosine-specific kinases of viral origin revealed a significant degree of sequence identity among all of these proteins. These data suggest that calcium-dependent protein kinases may share a common ancestral gene and a common structural basis for catalytic function with a wide variety of other protein kinases which respond to different signals and control quite different processes.

Amino acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3',5'-phosphate dependent protein kinase.

The complete amino acid sequence of the regulatory subunit of type I cAMP-dependent protein kinase from bovine skeletal muscle is presented. The S-carboxymethylated protein was cleaved with cyanogen bromide to provide a complete set of nonoverlapping fragments. These fragments were overlapped and aligned by using peptides generated by proteolytic cleavage. The protein contains 379 amino acid residues corresponding to a molecular weight of 42 804. As in the type II regulatory subunit of cAMP-dependent protein kinase, a pattern of internal gene duplication is observed, which is consistent with two cAMP-binding domains. The two types of regulatory subunit from type I and type II kinase display similarities in domain substructure and in amino acid sequence, which provide a molecular basis for new insight into their regulatory roles. Detailed analyses of the homology of the regulatory subunits of type I and type II cAMP-dependent protein kinase and of similar relationships to cGMP-dependent protein kinase and Escherichia coli catabolite gene activator protein are presented in accompanying reports from this laboratory [Takio, K., Smith, S. B., Krebs, E. G., Walsh, K., & Titani, K. (1984) Biochemistry (second paper of three in this issue); Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry (third paper of three in this issue)].