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Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras Guy Bauw, Heidi V. Nielsen, Jeppe Emmersen, Ka ˚ re L. Nielsen, Malene Jørgensen and Karen G. Welinder Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark Potato (Solanum tuberosum) is the world’s fourth most important crop after rice, wheat and corn. Originating in South America, this plant is now cultivated and consumed worldwide. The potato tuber is also an industrially important source of starch. Centuries of breeding have resulted in thousands of variants adap- ted to specific climatic, agricultural, pest resistance, nutritional, sensory and industrial requirements. Despite the importance of the potato tuber for the food and starch industries, detailed knowledge of potato tuber proteins is scarce. Potato tuber proteins are classified into three groups: patatins, protease inhibitors, and other proteins [1]. The patatin and pro- tease inhibitor classes, which form the bulk of the potato tuber protein, are mostly considered to be stor- age proteins. The majority of the isoforms have defined enzymatic and inhibitory activities that might be of physiological relevance. For example, several studies have indicated that purified patatin and certain protease inhibitors reduce the growth of larvae [2–5]. Potato tuber protease inhibitors are a diverse group of proteins that inhibit a variety of proteases and some other enzymes, for example invertase [6]. Some have dual or broad substrate specificity [4]. They differ in Keywords Kunitz protease inhibitor; patatin; Solanum tuberosum; lipoxygenase; cultivar markers Correspondence K. G. Welinder, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark Tel: +45 963 58467 E-mail: welinder@bio.aau.dk (Received 5 April 2006, revised 18 May 2006, accepted 7 June 2006) doi:10.1111/j.1742-4658.2006.05364.x The major potato tuber proteins of the Kuras cultivar, which is the domin- ant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis. The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanum tuberosum). Proteins were characterized using MS and scored against potato protein databases, derived from both ‘Kuras only’ and ‘all potato’ expressed sequence tags (EST) and full-length cDNAs. Despite the existence of  180 000 ESTs, the currently available potato sequence data showed a severe under-representation of genes or long tran- scripts encoding proteins > 50 kDa (3.5% of all) compared with the com- plete proteome of Arabidopsis thaliana (33% of all). We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras. Other proteins identified include annexin, glyoxalase I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants. Known S. tuberosum patatins cluster into three clades all represented in Kuras. S. tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPI K, found to date only in Kuras. Furthermore, protein abun- dance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber. Abbreviations EST, expressed sequence tag; IPG, immobilized pH gradient; KPI, Kunitz protease inhibitor; pI, isoelectric point; PMF, peptide mass fingerprint; SAGE, serial analysis of gene expression; St, Solanum tuberosum. FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS 3569 their amino acid sequence, chain length (M r 4, 8, and 20–22 kDa) and subunit composition (monomer to pentamer) [7]. According to the recently introduced general classification of protease inhibitors [8], potato tuber contains inhibitors of five nonhomologous super- families I3A, I13, I20, I25B and I37 (http://www. merops.ac.uk). By contrast to potato protease inhibitors, patatins constitute a more uniform group of homologous pro- teins. Pots et al. [9] separated the patatins of the Bintje cultivar into four pools with different chromatographic and electrophoretic characteristics, but similar biophys- ical properties. Patatins catalyse the nonspecific hydro- lysis of a wide range of acyl and phospholipids [5,10]. Genes encoding patatins [11,12], and Kunitz protease inhibitors (KPIs) [13–15] of different potato cultivars have been cloned and sequenced. Based on genetic and molecular analysis, Twell and Ooms [12] estimated that there are 64–72 patatin DNA copies in the tetraploid genome of potato cv. Desiree. Little information on other proteins present in the tuber is available, and until recently, no systematic gene discovery or protein sequencing had been under- taken. Only a few studies have reported on the protein content and its regulation upon physiological changes. The protein content of the potato tuber functioning as a sink during maturation has been compared with the tuber acting as a source for sprouting and the initi- ation of a new plant by Borgmann et al. [16]. Espen et al. [17] reported on protein changes upon tuber dor- mancy, whereas De ´ sire ´ et al. [18,19] used agar-grown microtubers to analyse protein changes due to tuberi- zation and the breaking of dormancy. None of these reports analysed the proteins of interest by sequencing. Recently, Lehesranta et al. [20] identified different pro- teins from the cultivar Desiree using LC-MS ⁄ MS, as part of an extensive comparison of the 2D gel elec- trophoretic protein profiles of different potato variants, and the influence of genetic engineering on the potato tuber protein profile. Potato gene discovery using expressed sequence tag (EST) sequencing [21–23] (http://www.tigr.org) has recently generated a large number of partial nucleotide sequences, originating from different potato cultivars and tissues. These partial sequences, together with the limited number of full-length cDNA sequences have been assembled in continuous nucleotide sequences or contigs, generating a plethora of potential protein sequences. The Kuras cultivar, having a superior pest resist- ance, is the major starch potato grown in Northern Europe. A large number of EST sequences expressed in the mature tuber are available for this particular cultivar [21], together with an increasing number of full-length cDNA sequences (HV Nielsen, KL Nielsen & J Emmersen, unpublished results). Furthermore, the transcriptome of the mature tuber cv. Kuras was recently analysed using serial analysis of gene expres- sion (LongSAGE), which generated 19 nucleotide sequence tags of the expressed genes [24]. Here we report on electrophoretic separations of Kuras tuber proteins, the protein chemical characterization of the major proteins and the classification of currently known patatins and KPIs into clades based onto sequence similarity. This study provides clear evidence that the protein variants within the three distinct clades of patatins pat1, pat2 and pat3, and the five clades of Kunitz protease inhibitors, KPI A, KPI B, KPI C, KPI K (Kuras), KPI M (miraculin-like) expressed in Kuras tuber, are cultivar specific. Moreover, to date, KPI K has been found only in cv. Kuras. In contrast, the amino acid sequences of other major proteins such as annexin, glyoxalase I, enolase, and lipoxygenase showed little, if any, sequence variation among potato variants. Results Electrophoretic patterns of potato tuber proteins from cv. Kuras 2D-PAGE Protein was extracted from mature field-grown potato tubers (S. tuberosum cv. Kuras) and separated using 2D-PAGE, using a broad range immobilized pH gradi- ent (IPG) pH 3–10 as the first dimension (Fig. 1). Vis- ual inspection of the protein patterns of individual tubers did not reveal major differences. The number of individual protein spots detectable is limited as two protein groups make up the majority of tuber proteins in Kuras, the overlapping patatins with a molecular mass of 40–45 kDa, isoelectric point (pI) 4–5, and the KPI with a molecular mass of 20–22 kDa spread over the entire pI range. To improve the resolution, proteins were separated on IPG strips, pI 4–7 and 6–11. On a typical 2D gel, pI 4–7, developed using Coomassie Brilliant Blue, epi- cocconone or silver staining, 250, 550 and 600 protein spots were detected, respectively (supplementary Fig. S1). In general, a four- to eightfold higher amount of protein was applied to 2D gels stained with Coomassie Brilliant Blue and used for protein analyses (Fig. 2). Apart from the major proteins at 20–22 and 40– 45 kDa, few proteins are seen in Coomassie Brilliant Blue-stained gels, even after enhancement with computer software. Staining with the more sensitive Potato tuber proteins G. Bauw et al. 3570 FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS epicocconone or silver stains revealed more spots dis- tributed throughout the pI and M r ranges. On a Coomassie Brilliant Blue-stained gel (pI 6–11) only a few well-defined protein spots at 20–22 kDa were visible. Epicocconone or silver staining revealed 300 and 280 protein spots, respectively, most on the neutral side of the 2D gel (supplementary Fig. S1). The intensity and the area of the minor protein spots were at least two orders of magnitude lower than the abundant proteins visible using Coomassie Brilliant Blue staining (Fig. 2). Unmodified versus reduced and alkylated 2D-PAGE protein patterns Figures 1 and 2 show unmodified tuber protein (i.e. without reduction and alkylation at any stage of the preparation), which we found most reproducible, and which retained disulfide-linked subunits in one protein spot. Reduction and alkylation, which have been used in most studies, resulted in altered 1D and 2D patterns (Fig. 3, supplementary Fig. S2). The most remarkable changes were observed for the 20–22 kDa KPIs. Redu- cing the IPG-focused protein by including 50 mm dithiotreitol in the SDS equilibration buffer before 2D electrophoresis, changed the position of some protein spots. This indicates a more complete unfolding due to disulfide bond cleavage (decreased mobility), or clea- vage of disulfide-linked subunits (increased mobility). When the reduction was performed before any elec- trophoretic separation, both pI and size changed for the same reasons. Additional alkylation of cyste- ines using the neutral iodoacetamide (supplementary Fig. S2D) should look similar to reduction only (sup- plementary Fig. S2C), but showed less-distinct spots for unknown reasons. Alkylation with iodoacetic acid introduces negative charges at all cysteines, resulting in lower pI values. This shifted almost all 20–22 kDa pro- teins to the pI 4–7 region (supplementary Fig. S2E). Tuber proteins of molecular masses 50–120 kDa Protein spots with higher molecular masses varied in intensity from 2D gel to 2D gel, probably due to restricted entry into the polyacrylamide network. Fur- thermore, because many of these proteins are resolved in spot series of identical mass, the tuber proteins were separated by 1D SDS ⁄ PAGE using a cross-linking of 0.5% instead of 2.6% to improve protein yield, separ- ation and reproducibility (Fig. 3). The intensities of the individual protein bands in the SDS gel provided an estimate of their relative abundance. The mobility of proteins changed upon reduction of disulfide bridges as seen by comparing lanes 2 and 3 of Fig. 3. Identification of potato tuber proteins Thirty-nine unmodified protein spots from Coomassie Brilliant Blue-stained 2D gels (Fig. 2) were assigned using tryptic peptide mass fingerprint (PMF) analysis Mr 3pI 10 66 45 35 25 18 (kDa) Fig. 1. Broad pI range 2D gel of potato (cv. Kuras) tuber proteins. Protein was extracted from potato tuber and separated by isoelec- tric focusing IPG pH 3–10 in the first dimen- sion and by 12.5% SDS ⁄ PAGE in the second dimension. One milligram of unmodified protein (i.e. not reduced or modified) was applied, and the proteins stained using epicocconone. G. Bauw et al. Potato tuber proteins FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS 3571 and curated manually (Table 1). Fifteen reduced and alkylated potato tuber protein spots were also identi- fied by PMF (data not shown), and confirmed the data given in Table 1. S. tuberosum patatins The prominent acidic protein spots of 40–45 kDa were patatin variants (Table 1, Fig. 4). Kuras EST patatin sequences were assembled into eight contigs and two singletons [25] (http://www.bio.aau.dk/en/st-data.htm), and were confirmed using full-length sequencing of seven different clones (GenBank accession numbers DQ114415–DQ114421). The phylogenetic tree based on the patatin cDNA sequences (Fig. 5) showed that the Kuras patatin genes cluster in three clades, pat1 with the Kuras-specific pat1-k1, -k2 variants, pat2 which is more diverse including Kuras-specific pat2-k1, -k2, -k3, -k4 variants, and pat3 with only a single form in Kuras, pat3-k1. The patatin sequences showed pronounced cultivar-dependent variation among the currently known potato patatin genes (Fig. 5) and proteins (Fig. S3). Kuras patatins are 84–96% identical in terms of their amino acid sequences, and have 80– 95% identity with published potato patatin sequences from various strains. The higher mass patatin spots 27–31 (Fig. 2) were identified as pat3-k1, which contains three potential N-glycosylation sites, two of which were confirmed using PMF analysis (Fig. 4). This prominent patatin Mr (kDa) 66 45 35 25 18 25 611pI 3 A B C 7pI 18 Fig. 2. Annotation of the protein spots on Coomassie Brilliant Blue- stained gels. (A) 2D gel pI 4–7 of potato tuber proteins. (B) Enlarge- ment of patatin spots boxed in gel (A). (C) Lower half of the 2D gel pI 6–11 of potato tuber. Mr (kDa) 1 2 3 SP1 Lip Eno Pat 200 150 120 100 85 70 60 50 40 30 Fig. 3. SDS ⁄ PAGE separation of high molecular mass potato tuber proteins stained with Coomassie Brilliant Blue. Lane 1, molecular mass markers; lane 2, protein reduced with dithiotreitol and alkylated with iodoacetic acid; lane 3, unmodified protein extract. Predomin- ant identified proteins of lane 3 are indicated by arrows, starch phosphorylase 1 (SP1), lipoxygenase (Lip), enolase (Eno) and pata- tin (Pat). Potato tuber proteins G. Bauw et al. 3572 FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS Table 1. Identification of 2D gel-separated tuber proteins from mature field-grown potato (cv. Kuras). Spot Protein Accession number Matching peptides Sequence a coverage (%) Experimental Calculated a pI b M r (kDa) pI M r (kDa) Acidic proteins pI 4–7: 1 KPI M-k1 TC112274 c, d 10 34 6.0 20 6.08 21.2 2 KPI K-k1 TC112888 d 13 64 5.0 20 5.06 20.1 3 KPI M-k2 TC112554 d 5 26 4.67 22 5.21 21.7 4 KPI B-k1 DQ168319 e 10 58 4.47 21 5.18 20.4 5 KPI M-k2 TC112554 d 6 30 4.05 23 5.21 21.7 6 KPI B-k2 DQ168331 e 12 72 3.70 19 6.12 20.3 7 KPI B-k1 DQ168319 e 9 63 3.60 19 5.18 20.4 8 Unknown TC121765 d 5 24 4.00 29 9.90 27.6 9 KPI A-k1 DQ168311 e 10 65 5.50 21 8.99 20.8 10 KPI B-k3 DQ268836 e 12 59 5.81 21 6.36 20.0 11 KPI K-k1 TC112888 d 13 61 6.0 20 5.06 20.1 12 Unknown TC121765 d 7 29 4.47 35 9.90 27.6 13 Glyoxalase I TC119268 d 11 38 4.61 38 5.35 32.8 14 Annexin p34 TC119057 d 20 56 4.73 38 5.26 35.8 15 Patatin pat2-k3 DQ114419 e 14 55 5.1 45 5.40 40.0 16 Patatin pat2-k3 DQ114419 e 18 63 4.87 44 5.40 40.0 17 Patatin pat1-k1 DQ114415 e 14 51 4.82 49 5.26 40.1 18 Patatin pat1-k2, pat2-k1 DQ114416, e DQ114417 e 22 55 4.67 44 19 Patatin pat1-k1 DQ114415 e 22 54 4.58 45 20 Patatin pat1-k2, pat2-k1 ⁄ k3 24 51 4.39 44 21 Patatin pat1-k2, pat2-k1 ⁄ k3 22 61 4.30 44 22 Patatin pat1-k2, pat2 17 52 4.17 43 23 Patatin pat1-k2, pat2 20 55 4.06 43 24 Patatin pat1-k2, pat2-k2 21 51 3.96 43 25 Patatin mix 20 52 3.87 43 26 Patatin mix 20 50 3.80 44 27 Patatin pat3-k1 DQ114421 e 21 86 3.9 47 4.76 40.0 28 Patatin pat3-k1 DQ114421 e 23 86 3.82 47 4.76 40.0 29 Patatin pat3-k1 DQ114421 e 13 71 3.74 47 4.76 40.0 30 Patatin pat3-k1 DQ114421 e 16 71 3.66 47 4.76 40.0 31 Patatin pat3-k1 DQ114421 e 14 63 4.22 50 4.76 40.0 32 Enolase TC112026 d 9 33 4.72 56 5.49 47.8 Basic proteins pI 6–11: 61 KPI B-k4 K2-01917 f 11 44 6.0 21 6.36 20.0 62 KPI C-k1 K2-01866 f 5 36 7.8 23.0 7.72 20.2 63 KPI C-k1 K2-01866 f 6 40 7.8 22.5 7.72 20.2 64 KPI C-k2 K2-01879 f 8 41 8.4 22.3 9.43 20.0 65 KPI C-k2 K2-01879 f 8 41 8.6 22.1 9.43 20.0 66 KPI C-k3 K2-01896 f 6 40 8.4 21.0 9.25 20.1 67 KPI C-k3 K2-01896 f 6 40 8.4 20.0 9.25 20.1 68 KPI C-k2 K2-01879 f 8 41 8.7 22.3 9.43 20.0 69 KPI C-k3 K2-01896 f 10 57 8.7 21.0 9.25 20.1 a Mature protein (removal of signal sequence and known pro-peptides). b pI predicted with cystine (SS) using GPMAW [46]. c Tentative contig number with best match. Kuras proteins deviate at certain positions. d TIGR accession numbers at http://www.tigr.org/tigr-scripts/tgi/ T_index.cgi?species=potato. Tentative contigs might be > 95% identical to Kuras-specific sequence. e GenBank accession of full-length cDNA sequence of Kuras (HV Nielsen, KL Nielsen & J Emmersen, unpublished results). f Kuras contigs accession number at http:// www.bio.aau.dk/en/st-data.htm. G. Bauw et al. Potato tuber proteins FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS 3573 has most likely lost its lipolytic activity due to a Ser to Gly substitution (position 54) (Fig. 4) in the proposed catalytic centre [26], which supports its role as a stor- age protein. Protein spots 15 and 16, pat2-k3, and 17 and 19, pat1-k1, seemed to be single patatins (Table 1). The MS spectra of the remaining patatin spots 18 and 20–26, contained tryptic peptide mass values derived from different Kuras patatin variants, as they included masses of homologous peptides from two variants (e.g. spot 18 contained pat1-k2 and pat2-k1, Fig. 4). None of the potential N-glycosylation site(s) of the pat1 and pat2 variants were verified by tryptic peptides. Although pat1-k1 contains two potential N-glycosyla- tion sites, one in common with pat3-k3, the remaining patatins have only a single potential site. All isopatatin spots 15–31 showed a mass peak of 1705.9 ± 0.2 Da corresponding to their identical N-terminal peptides, TLGEMVTVLSIDGGGIK (Table 2). The unique N-terminal peptide of pat1-k2 was not seen in spot 18 (Table 2). An overview of the patatin results are shown in Fig. 4. S. tuberosum Kunitz protease inhibitors (StKPI) The prominent 20–22 kDa proteins were distributed over the entire pI range belong to the KPI family, which includes the classical soybean trypsin inhibitor. Our results are best presented in the context of a con- sistent nomenclature based on sequence similarity. The protein sequences of KPIs in various plants, including Arabidopsis and tomato, contain the 17-residues KPI signature [LIVM]-x-D-{EK}-[EDHNTY]-[DG]- [RKHDENQ]-x-[LIVM]-x-{E}-x-x-x-Y-x-[LIVM], where H is observed in some potato miraculin-like seq- uences ([], residues allowed, and {}, residues excluded from the position; X, any residue) (http://www.expasy. org/cgi-bin/nicedoc.pl?PDOC00255) [27]. In Kuras tubers Fig. 4. Alignment of amino acid sequences of the mature patatin proteins of potato cv. Kuras using pat1-k1 as template. (.) Identical to tem- plate; (–) gap. The single Cys162 conserved in all patatins, and the catalytic Ser54 substituted to Gly in pat3-k1 are shown in bold. Potential N-glycosylation sites are shown in small italics or bold capitals, if verified by PMF. The predominant potato protein N-linked glycan [48] (Xyl)Man 3 (Fuc)(GlcNAc) 2 gives rise to a mass increase of 1171 Da. Grey shaded residues were covered by peptide masses of the PMF ana- lysis of a particular 2D spot, pat1-k1 ¼ spot 19, pat2-k3 ¼ spot 16, pat3-k1 ¼ spot 28. Both pat1-k2 and pat2-k1 were present in spot 18. Underlined sequences were identified by nanospray MS ⁄ MS analysis. Corresponding GenBank accession numbers are found in Table 1. Potato tuber proteins G. Bauw et al. 3574 FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS we found five StKPI clades represented (Figs 6,7; sup- plementary Fig. S4): KPI A, KPI B, KPI C, in accord- ance with the nomenclature of Ishikawa et al. [14] (cv. Danshaku) and Heibges et al. [15] (cv. Provita and cv. Saturna); KPI K (to date found only in Kuras); and KPI M, similar to the sweet tasting miraculin des- cribed by Theerasilp et al. [28]. Thirteen Kuras KPI sequences of the KPI A, B, and C clades were subjected to full-length cDNA sequencing (GenBank accession numbers DQ168311, DQ168316–DQ168319, DQ168324, DQ168325, DQ168327, and DQ168329–DQ168333). Some of these sequences differ only outside the reading frame. Ku- ras-specific contigs and tentative contigs, for all known potato ESTs are available at http://www.bio.aau.dk/ en/st-data.htm and http://www.tigr.org, respectively. Full-length cDNA sequences, a contig for KPI K (K2- 01900, TC112888), and two contigs for KPI M (K1- 01724, TC112274 and TC112554) may account for the tryptic peptide data of protein spots 1–7, 9–11, and 61–69 (Table 1, Fig. 6). Masses verifying the N- and C-termini of mature KPIs were observed for variants of the KPI A, KPI B and KPI C clades (Table 2, Fig. 6). The 2D gels show that KPI C variants all have basic pI values of 7.0–8.6, whereas KPI A, KPI B, KPI K and KPI M variants are acidic to neutral with experi- mental pI values of 4.0–6.0 (Fig. 2). Analysis of StKPI sequences showed pronounced cultivar-dependent variation among the known genes (Fig. 7) and proteins (Fig. S4). The amino acid identity among variants within each of the five clades is > 83% for Kuras extending to 77% including all culti- vars, whereas the StKPI interclade identity varies from 75 to 15%. Therefore, StKPIs constitute a much more diverse protein family than St patatins. Other major proteins In addition to the dominating patatins and KPIs in Kuras, five other proteins, annexin, glyoxalase I, eno- lase, and two of unknown function were identified from the 2D-gel (Table 1; Fig. S5). Masses correspond- ing to acetylated N-termini of mature annexin and gly- oxalase I, together with their C-termini were observed in the MS spectra (Table 2). The 50–120 kDa region of reduced tuber protein separated by 1D SDS ⁄ PAGE (Fig. 3, lane 2) was cut into successive 10 · 2 mm slices from an unstained part of the gel, digested with trypsin and analysed, whereas discrete protein bands were cut from unmodi- fied protein (Fig. 3, lane 3). The mass spectra of the 1D samples show a higher background level of low- intensity ions from minor proteins as expected. There- fore, a total of 70–120 intense and well-defined mass values were collected from the nine mass spectra of each gel band. Fourteen different proteins were identi- fied unambiguously by at least 15 tryptic peptide masses, most in 2–4 adjacent gel slices (Table 3; sup- plementary Table S1). In contrast to the patatin and KPI protein families, our extensive analysis of the amino acid sequences of other major proteins, i.e. glyoxalase I, annexin, eno- lase, catalase, UTP : glucose 1-phosphate uridyltrans- ferase (UDP pyrophosphorylase) (supplementary Fig. S5), revealed no or only very limited (< 2%) sequence variation among S. tuberosum strains. Discussion Tuber proteins from mature field grown Kuras pota- toes were characterized using 1D and 2D gel electro- phoresis, and the major proteins identified by peptide mass fingerprinting. Unmodified protein provided the most reproducible 2D pattern. We show how the gel Fig. 5. Phylogenetic tree of S. tuberosum patatin cDNA sequences. The tree was constructed using minimum evolution dis- tance analysis. The corresponding protein sequences and refer- ences are shown in supplementary Fig. S3. Bootstrap values of 1000 resamplings are indicated at the nodes of the tree. Non-Kuras patatins genes are indicated with their GenBank accession num- bers. Scale bar indicates five substitutions per 1000 nucleotides. G. Bauw et al. Potato tuber proteins FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS 3575 electrophoretic patterns of potato tuber proteins change with common chemical treatments such as disulfide reduction and alkylation. The rather high sequence coverage obtained in this study was essential to the successful distinction among the many cultivar specific patatin and KPI variants. Separation of the tryptic peptide mixtures using stepwise elution from Poros beads combined with the use of different MALDI matrices [29,30] provided this high coverage. For some proteins, the maximum possible sequence coverage was reached, because missing sequences were either small hydrophilic tryptic peptides (fewer than seven residues) or long hydrophobic ones with low recoveries from Poros beads and masses outside the MALDI-reflectron TOF window. Nanospray MS ⁄ MS analyses provided only limited additional information (Figs 4, 6). N- and C-terminal tryptic peptides of mature proteins were accounted for in several cases (Table 2). Patatins Seven patatins have been cDNA sequenced and also identified as proteins in mature Kuras tuber. All known S. tuberosum patatins are distributed into three clades based on sequence similarity, and those of Kuras are cultivar dependent. Also the relative abundance of patatin in tubers shows a marked dependence on cultivar [1,31,32]. The statistical analy- sis of the abundance of 2D gel separated proteins of > 20 potato cultivars, landraces and genetically modi- fied potatoes by Lehesranta et al. [20] demonstrated a high variability of patatins in general. Comparing the similarly prepared tuber protein and 2D gels of Kuras and of Desiree, Maris Piper and a landrace of Lehes- ranta et al. [20], the patatin content in Kuras is remarkably high. Comparing total protein distribution of some common Danish potato varieties by SDS ⁄ PAGE supported this greater abundance of patatins in Kuras (M. Jørgensen & K.G. Welinder, unpublished results). Kunitz protease inhibitors Protein reduction changed the pattern of KPIs signifi- cantly, due to the cleavage of two conserved disulfide bonds [33], at positions Cys88–Cys141 and Cys197– Cys214 (Fig. 6). In fact, KPI clades show an extensive variability within the last cystine loop. KPI A has an insertion, which is present at both the nucleotide and protein levels. KPI B has a variable insertion at the nucleotide level. When present, it is removed as a pro- peptide at the protein level. This gives rise to separ- ation into a larger N-terminal and a smaller C-terminal subunit of KPI B after reduction (supple- mentary Fig. S2). The two-chain structure of this type Table 2. Masses of the N- and C-terminal tryptic peptides of 2D gel proteins. All experimental masses deviated < ± 0.2 Da from the calcu- lated monoisotopic masses. Spot Protein Sequence of tryptic peptide Calculated mass (Da) [M + H] + Ref. N-terminal peptides: 4,7 KPI B-k1 LPSDATPVLDVTGK 1412.76 [33] 6 KPI B-k2 LPSDATPVLDITGK 1426.77 [33] 9 KPI A-k1 ESPLPKPVLDTNGKELNPNLSYR 2581.35 [50] 10 KPI B-k3 LPSDATPVLDVTGK 1412.76 [33] 13 Glyoxalase I ac-AEASAPAVPSTELLEWPKKDKR a 2465.30 14 Annexin p34 ac-ASLTVPAEVPSVAEDCEQLR a 2156.00 15–31 Patatin TLGEMVTVLSIDGGGIK b 1705.89 [9] 61 KPI B-k4 LPSDATPVLDVTGK 1412.76 [33] 62,63 KPI C-k1 LVLPEIYDRDGDPLR 1770.93 [51] 64,65,68 KPI C-k2 LVLPEVYDQDGEPLR 1742.89 [51] 66,67,69 KPI C-k3 LVLPEVYDQDGHPLR 1750.90 [51] C-terminal peptides: 2,11 KPI K-k1 VALVSNFSLDFEFEKVED 2088.01 9 KPI A-k1 RLALVNENPLDVLFQEV 1969.07 [50] 13 Glyoxalase I TVLVDNDFLKELESK 1878.02 14 Annexin p34 DTGGDYENMLVALLGQEEE b 2098.90 61 KPI B-k4 DNPLDVSFQVQ 1389.69 [33] 64,65,68 KPI C-k2 LAAVDDDKDFIPFVFIKA 2024.07 [51] 66,67,69 KPI C-k3 LVTVDDDQDFIPFVFIKA 2082.07 [51] a ac, acetyl (+ 42.0 Da). b Met detected as methionine sulfoxide. Potato tuber proteins G. Bauw et al. 3576 FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS of KPI was first documented by Valueva et al. [33] using complete amino acid sequencing. KPI C and KPI M (miraculin) have no inserts at the nucleotide level, and produce continuous peptide chains on reduc- tion, similar to KPI A. KPI K has the nucleotide insert. The recognition of a peptide ion mass of 2197.15 covering the sequence LLGYELITCD- GALVTMGQR (Cys-acrylamide, methionine sulfox- ide) indicates that this insert might be retained in the mature protein, although unambiguous verification is required. KPI K is unique to Kuras so far and was represented by 24 ESTs (TC112888; K2-01900) [21]. It will need further structural and functional characteri- zation. Contrasting with KPIs, patatins changed only on carboxymethylation due to the presence of a single cysteine only (Cys162) (Fig. 4). Fig. 6. Alignment of Kunitz protease inhibitors identified in Kuras potato tubers. (.) Identical to template residue of the KPI clade; (–) gap; (italics) ER signal sequence predicted by SIGNALP [49]. Cystein residues are shown in bold. Cys88–Cys141 and Cys197–Cys214 are expected to form disulfide bridges [33]. Grey shaded residues were covered by the masses of the PMF analysis of a particular 2D spot, KPI A-k1 ¼ spot 9, KPI B-k1 ¼ spot 4, KPI B-k2 ¼ spot 6, KPI B-k3 ¼ spot 10, KPI B-k4 ¼ spot 6, KPI C-k1 ¼ spot 63, KPI C-k2 ¼ spot 68, KPI C-k3 ¼ spot 69, KPI K-k1 ¼ spot 11, KPI M1 ¼ spot 1, KPI M2 ¼ spot 5. Underlined sequences were identified by nanospray MS ⁄ MS analysis. Corresponding GenBank accessions are found in Table 1. The 17 residues Kunitz motif is boxed by a solid line. The highly variable, absent or split sequence in KPIs is boxed by a dotted line. G. Bauw et al. Potato tuber proteins FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS 3577 Potato protein databases The identification of potato tuber proteins of 50– 120 kDa was limited due to their variable intensities in 2D gels, and due to the present severe under-represen- tation of long cDNAs and contigs among  180 000 ESTs. Thus the assembled TIGR tentative contigs cod- ing for potato proteins with more than 450 amino acid residues constituted only 3.5% (1314 sequences) of all, whereas this is 33% (± 12 000 sequences) for the complete Arabidopsis thaliana proteome. Also potato proteins 30–50 kDa predicted from EST contigs accounting for 12% (4465 sequences) of all were under-represented compared with the size distribution within the A. thaliana proteome (33%). To reduce the occurrence of false positives, identification of 1D gel separated proteins was presently restricted to those with 15 or more matching masses, sequence coverage of 25% or more, and identification in at least two adjacent gel slices (supplementary Table S1). A further limitation of this study was the absence of small pro- teins 3–10 kDa in the protein gels, such as carboxy- peptidase inhibitors, which can be obtained by chromatography [7]. Potato tuber proteome versus transcriptome The protein ensemble of tuber is of interest to the potato starch industry, as predominant proteins might be purified in quantity from potato juice, a waste from Fig. 7. Phylogenetic tree of S. tuberosum KPI cDNA sequences. The tree was con- structed similarly to Fig. 5. The correspond- ing protein sequences and references are shown in supplementary Fig. S3. Potato tuber proteins G. Bauw et al. 3578 FEBS Journal 273 (2006) 3569–3584 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... [34] Cultivar-specific sequences The patatins and protease inhibitors are generally considered to be the storage proteins of potato tuber, although most patatin variants (pat1 and pat2) have broad lipase activity [5,10,26,31], and the potato protease inhibitor variants have broad and highly variable specific activities against Ser-, Cys-, Asp-, or metalloproteases [7,35,36] In addition, both protein... roles in pest and insect resistance [2–5] Therefore, the exceptional expression and sequence variability within the protein clades of patatins and Kunitz inhibitors among potato variants is compatible with their dual roles in protein storage and resistance The accelerated molecular evolution has been driven by plant breeding The cultivar-specific nucleotide ⁄ amino acid sequences of patatins and KPIs, in... (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens Annu Rev Phytopathol 28, 425–449 3 Strickland JA, Orr GL & Walsh TA (1995) Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers Plant Physiol 109, 667– 674 4 Birk Y (2003) Plant Protease Inhibitors: Significance in Nutrition, Plant Protection, Cancer Prevention and. .. Hattori T & Nakamura K (1994) A family of potato genes that encode Kunitz- type protease inhibitors: structural comparisons and differential expression Plant Cell Physiol 35, 303– 312 15 Heibges A, Glaczinski H, Ballvora A, Salamini F & Gebhardt C (2003) Structural diversity and organization of three gene families for Kunitz- type enzyme inhibitors from potato tubers (Solanum tuberosum L.) Mol Genet Genomics... M-k1 (0.36%), KPI K-k1 (2.41%) and KPI B-k1, -k3 and -k4, which share a single SAGE tag (7.27%) Hence, the relative 2D gel protein intensities and SAGE tag abundance among patatin variants and among KPI variants appear remarkably similar and uniform in mature Kuras tuber However, in general, the range of protein expression is much wider than the range of mRNA abundance, and exceeds the limited dynamic... in 3 mL extraction buffer and centrifuged The combined supernatants were thoroughly mixed with 5 mL water-saturated phenol, incubated for 30 min on ice, and the two phases were separated by centrifugation (8000 g, 10 min) The upper phenol phase was removed and kept separately The intact interphase and the water phase were extracted with another 4 mL of water-saturated phenol and centrifuged Protein was... removed, and 100 lL of H2O added The tryptic peptides adsorbed to the beads were then stored at )20 °C until analysis MS analyses MALDI-TOF MS was performed on a Reflex III (Bruker Daltonics, Bremen, Germany) set at an initial acceleration Potato tuber proteins voltage of +20 kV and operated with pulsed ion extraction and in reflectron mode Laser intensity and reflectron were adjusted for optimal resolution and. .. Glaczinski H, Heibges A, Salamini R & Gebhardt C (2002) Members of the Kunitz- type protease gene family of potato inhibit soluble tuber invertase in vitro Potato Res 45, 163–176 7 Pouvreau L, Gruppen H, Piersma SR, van den Broek LAM, van Koningsveld GA & Voragen AGJ (2001) Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv Elkana J Agric Food Chem 49, 2864–2874... post-translational modifications such as cleavage of presequences and glycosylation, and inclusion of common chemical modifications, i.e acrylamidation of Cys and oxidation of Met, Trp and Cys Bioinformatic analysis Multiple alignments (protein settings: similarity matrix identity, gap initiation penalty ¼ 8, gap extension penalty ¼ 2) and phylogenetic analyses (minimum evolution distance analysis, bootstrap... G et al (2005) Potato expressed sequence tag generation and analysis using standard and unique cDNA libraries Plant Mol Biol 59, 407–433 Nielsen KL, Grønkjær K, Welinder KG & Emmersen J (2005) Global transcript profiling of potato tuber using LongSAGE Plant Biotech J 3, 175–185 Emmersen J (2002) Tools for EST and full cDNA sequencing of plants and analysis of the associated biological information PhD . Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras Guy Bauw, Heidi V. Nielsen, Jeppe Emmersen, Ka ˚ re L. Nielsen, Malene Jørgensen and Karen. patatin and certain protease inhibitors reduce the growth of larvae [2–5]. Potato tuber protease inhibitors are a diverse group of proteins that inhibit a variety of proteases and some other enzymes,. patatins [11,12], and Kunitz protease inhibitors (KPIs) [13–15] of different potato cultivars have been cloned and sequenced. Based on genetic and molecular analysis, Twell and Ooms [12] estimated