Gregory M. Neumann

Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin.

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6+/-0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma-thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC(50) 2 µM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor.

Purification and characterization of basic proteins with in vitro antifungal activity from seeds of cotton, Gossypium hirsutum

Abstract Basic proteins from seeds of cotton (Gossypium hirsutum) were purified by a procedure involving cation exchange chromatography on carboxymethyl cellulose (CM52) and reversed phase high performance liquid chromatography (HPLC). The basic protein fraction from these seeds contains a multiplicity of vicilin-derived 9–11 kDa proteins, a 16.3 kDa protein that has an N-terminal sequence nearly identical to that of a cotton cysteine-rich small vicilin protein and a 46.3 kDa protein having an N-terminal sequence with 61% identity and 75% similarity to the cDNA-based sequence of a γ-conglutin from Lupinus angustifolius. Edman N-terminal sequencing, electrospray ionization mass spectrometry (ESMS) and literature cDNA-based sequencing data were used to define the sequences of the vicilin-derived proteins. The inferred N- and C-terminal sequences of the 9–11 kDa proteins correspond to N-terminally truncated forms of cysteine-rich small vicilin proteins (such as the 16.3 kDa protein) suggesting that both could be processed from the vicilin (α-globulin) preproprotein. The various cotton basic proteins were found to have selective growth inhibitory activity in vitro against the filamentous fungi Botrytis cinerea, Alternaria brassicicola, Chalara elegans and Fusarium oxysporum. These proteins differ, however, from numerous other seed antifungal proteins in being neither substrates nor inhibitors of signal transduction elements such as wheat germ Ca2+-dependent protein kinase (CDPK), rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK), rat brain Ca2+- and phospholipid-dependent protein kinase (PKC) and chicken gizzard calmodulin-dependent myosin light chain kinase (MLCK).

Purification and sequencing of a family of wheat lipid transfer protein homologues phosphorylated by plant calcium-dependent protein kinase

Four low molecular weight, basic proteins (WBP1A, WBP1B, WBP2 and WBP3) that are substrates for wheat germ Ca(2+)-dependent protein kinase (CDPK) were purified from wheat germ by a procedure involving batchwise cation exchange on carboxymethylcellulose (CM52), acid precipitation, cation exchange HPLC on an SP5PW column and reverse-phase HPLC on a C18 column. While WBP1A, WBP1B and WBP3 are phosphorylated by wheat germ CDPK exclusively on Ser residues, WBP2 is phosphorylated on both Ser and Thr residues. CDPK-catalysed phosphorylation sites on WBP1A and WBP1B were determined. With all four proteins the phosphorylated form comigrates with non-phosphorylated protein (Mr about 9 kDa) on SDS-PAGE. Average molecular masses of reduced WBP1A, WBP1B, WBP2 and WBP3 measured using electrospray ionisation mass spectrometry (ESMS) are 9389 Da, 9274 Da, 9479 Da and 9467 Da, respectively. The complete amino-acid sequences of WBP1A and WBP1B (determined by Edman sequencing and ESMS of proteolytically derived fragments) and N-terminal sequences of WBP2 and WBP3 are highly homologous to each other and to sequences of low molecular weight, basic plant lipid transfer proteins (LTPs).

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.

Purification, characterization and sequencing of a family of Petunia petal lipid transfer proteins phosphorylated by plant calcium-dependent protein kinase

Abstract A number of small basic proteins (Pet1, Pet2, Pet3, Pet4 and Pet5) were purified to homogeneity from petals of petunia (Petunia hybrida var. Old Glory Blue) by a procedure involving batchwise cation exchange chromatography on carboxymethyl-cellulose (CM52) and cation exchange HPLC on a sulphopropyl-based SP5PW column. Pet1 to Pet5 were identified from extensive N-terminal sequencing as having a high degree of sequence homology to each other and to other plant phospholipid transfer proteins. Pet1, Pet2, Pet4 and Pet5 are phosphorylated by wheat embryo Ca2+-dependent protein kinase (CDPK) whereas Pet3 is a very poor substrate for this enzyme. After tryptic digestion of [32P]phosphoPet1 and [32P]phosphoPet2 phosphorylated by CDPK and reversed phase HPLC-based purification of [32P]phosphopeptides, Edman sequencing of the purified [32P]phosphopeptides revealed major Ser phosphorylation sites of S40 and S70 in the sequences SQAS40TTP and GLPS70TCG in Pet1 and Pet2, respectively. These phosphorylation site sequences differ from the Basic-X-X-Ser(Thr) motif found with many synthetic peptide substrates of plant CDPK but are similar to each other and to phosphorylation sites on some other CDPK substrates involving A G , S T and P residues. The complete primary structure of Pet2 (90 residues) has been determined by Edman sequencing and electrospray ionization mass spectrometry of the native Pet2 and of proteolytically-generated fragments. While Pet2(19–90) and Pet2(32–90) retain activity as CDPK substrates, all smaller fragments tested were inactive. A CDPK is present in Petunia petals. After in vivo labelling of Petunia petals with [32P]phosphate the major labelled protein resolved by SDS-PAGE has a mass of 9.4 ± 0.6 kDa, similar to the Mr of Pet1, Pet2 and Pet3.

Phosphorylation of barley and wheat phospholipid transfer proteins by wheat calcium-dependent protein kinase

Abstract Wheat (Triticum aestivum L.) germ Ca2+-dependent protein kinase (CDPK) phosphorylates a wheat basic protein (WBP) which is a putative phospholipid transfer protein and one of the best protein substrates yet found for wheat CDPK. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [32P]phospho WBP resolved two [32P]phosphopeptide peaks. Edman sequencing of one these established that WBP is phosphorylated by CDPK on S17 within the sequence A13PCISYA. WBP may also be phosphorylated at an additional site. Wheat CDPK also phosphorylates barley (Hordeum vulgare) lipid transfer protein (LTP1) and [32P]phosphoLTP1 exactly comigrates with LTP1 on SDS-PAGE. Unlike wheat WBP, LTP1 is a relatively poor substrate for wheat CDPK and is phosphorylated largely on threonine. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [32P]phosphopeptide resolved one [32P]phosphopeptide and subsequent Edman sequencing of this phosphopeptide showed that LTP1 is phosphorylated on T15 within the sequence K11PCLTYVQ. This phosphorylation site amino acid sequence motif differs from the Basic-X-X-Ser(Thr) motif found for a variety of synthetic peptide substrates of wheat germ CDPK but corresponds to the Pro-Cys-X-Ser/Thr phosphorylation site motif found for the phosphorylation by wheat germ CDPK of WBP, which is a homologue of barley LTP1. The phosphorylation sites determined for WBP and LTP1 are similar structurally and are within precisely aligned homologous sequences.

Phosphorylation of a plant protease inhibitor protein by wheat calcium-dependent protein kinase

Abstract Wheat germ ( Triticum aestivum ) Ca 2+ -dependent protein kinase (CDPK) phosphorylates soybean ( Glycine max ) Bowman-Birk trysin/chymotrypsin inhibitor protein (BBI) and [ 32 P]phospho BBI comigrates with BBI on SDS-PAGE. The phosphorylation of S 34 within the sequence RLNSCHS 34 ACK was determined from isolation of [ 32 P]-phospho-LNSCHSACK from tryptic digests of [ 32 P]-phosphoBBI and from subsequent analysis of the Edman degradation products. Confirmation of S 34 phosphorylation was obtained from similar analysis of phosphopeptides purified from the tryptic digest of 2-bromoethylamine-derivatised [ 32 P]phosphoBBI. This phosphorylation site differs from the Basic-X-X-Ser(Thr) phosphorylation site specificity previously found for synthetic peptide substrates of wheat CDPK. The adjacent S 31 of BBI, which does lie within a Basic-X-X-Ser motif, is not phosphorylated. Bovine serum albumin (BSA) is also phosphorylated by wheat germ CDPK on a site not involving a Basic-X-X-Ser motif, namely on S 468 within the sequence K 464 TPVSEK 470 . The site of phosphorylation of BBI by CDPK is located in an exposed central loop lying between the two anti-parallel β-sheet protease inhibitory domains. Two other CDPK protein substrates have phosphorylation site sequences of either Ser-X-X-Basic or Ser-X-Basic as found for BBI and BSA, respectively, suggesting that a C-terminal side basic residue could be a substrate recognition determinant for plant CDPK on some protein substrates.

Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca2+-dependent protein kinase

The basic protein fraction from seeds of castor bean (Ricinus communis L.) contains 4732 Da and 4603 Da proteins phosphorylated in vitro by plant Ca(2+)-dependent protein kinase (CDPK). These proteins, RS1A and RS1B respectively, were purified by cation-exchange HPLC (SP5PW column) and reverse-phase HPLC (C18 column) and identified as napin-like protein small chains by Edman sequencing and electrospray ionization mass spectrometry (ESMS). The other R. communis 4 kDa small chains (RS2A, RS2B, RS2C and RS2D) are not phosphorylated by CDPK and neither is the corresponding 7332 Da large chain (RL) that forms 1:1 disulfide-linked complexes with RS2(A-D). RS1A/B is one of the best substrates found for plant CDPK (K(m) = 1.8 +/- 0.8 microM). RS2(A-D) (but not RL or RS1A/B) strongly inhibit calmodulin (CaM)-dependent myosin light chain protein kinase (MLCK) (IC50 = 0.25 microM) and inhibit the Ca(2+)-dependent enhancement of dansyl-CaM fluorescence. The basic protein fraction from seeds of bitter melon (Momordica charantia) also contains napin-like proteins that are 1:1 disulfide-linked complexes of a small chain (MS1, MS2, MS3 or MS4) and a large chain (ML). The M. charantia small chains were purified and completely sequenced by Edman degradation and ESMS. M. charantia small chains MS1, MS2, and MS4 (but not MS3) are phosphorylated by CDPK to unit stoichiometry on S21 within the sequence R17SCES21FLR. The R. communis small chain RS1A is phosphorylated on S34 within the sequence R31QSS34SRR. Both of these phosphorylation site motifs are consistent with those found for other plant CDPK substrates.

Identification of the site on potato carboxypeptidase inhibitor that is phosphorylated by plant calcium-dependent protein kinase

Abstract Potato ( Solanum tuberosum ) carboxypeptidase inhibitor (PCI) is phosphorylated by wheat Ca 2+ -dependent protein kinase (CDPK). While the K m of PCI for CDPK is high, the rates of phosphorylation of PCI by plant CDPK are also very high in relation to phosphorylation of the histone III-S preparation, an excellent substrate for plant CDPKs. Analysis of intact PCI and of HPLC-resolved chymotryptic fragments of [ 32 P]phosphoPCI (phosphorylated by plant CDPK) by electrospray ionization mass spectrometry (ESMS) confirmed the sequences of three PCI variants, PCIA, PCIB and PCIC. PCIB (4276.75 Da), PCIA (4148.62 Da) and PCIC (3900.38 Da) have differing N-terminal residues, namely 27 , Ser 29 and Ser 30 of PCIC, PCIA and PCIB, respectively. This phosphorylatable Ser is located within the CWNSARTC sequence and is associated with a Ser-X-Basic motif found with a number of other plant CDPK phosphorylation sites. The phosphorylatable Ser is located on an exposed loop at a secondary point of interaction of PCI with carboxypeptidase.

Purification and sequencing of yellow mustard seed napin small and large chains that are phosphorylated by plant calcium-dependent protein kinase and are calmodulin antagonists

Abstract A multiplicity of small (S) and large (L) napin subunits were purified from yellow mustard (Sinapis alba L.) seeds by a protocol involving extraction, successive batch-wise cation exchange chromatography on carboxymethylcellulose (CM52), cation exchange HPLC on an SP5PW column and reversed phase HPLC on a C18 column. Initial cation exchange HPLC resolved 4 major zones of proteins (M1, M2, N1 and N2) that can be phosphorylated by plant Ca2+-dependent protein kinase (CDPK). Electrospray ionization mass spectrometry (ESMS) revealed that M1 and M2 are 6 kDa proteins, later identified as γ-thionin-related proteins. ESMS of fractions N1 and N2 revealed the presence of 14.5 kDa proteins identified as napin complexes, each composed of a single small subunit linked to a single large subunit and involving 4 disulphide linkages. The napin complexes (N1A, N1B, N1C, N1D, N2A, N2B and N2C) were disrupted and the constituent small subunits (S1, S2 and S3) and large subunits (L1A, L1B, L1C, L2A, L2B and L2C) were resolved by reversed phase HPLC and precise average molecular masses determined by ESMS. The small and large subunits have average molecular masses of about 4.4 kDa and 10.1 kDa, respectively. The masses of each napin complex can be precisely accounted for from the masses of the constituent subunits. Thus the major complex N2A (14 569 ± 3 Da) is evidently composed of S3 (4434.0 ± 1.5 Da) and L2A (10 142.5 ± 1.5 Da) and involves 4 disulphides (loss of 8.0 Da), the expected mass of S3 + L2A-8H being 14 569 ± 2 Da. The yellow mustard napin large chain L2A is phosphorylated by wheat CDPK on Ser60 within the sequence LQHVIS60RIY. The complete sequence of this and other large (and small) napin subunits were determined from Edman sequencing and/or ESMS data by comparison with published napin sequences. Yellow mustard seed CM52-binding fractions decrease the Ca2+-dependent fluorescence emission of dansyl-CaM and yellow mustard small and large chains inhibit CaM-dependent myosin light chain kinase (MLCK).