Proteomic Approach for Caudal Trauma-Induced Acute Phase Proteins Reveals That Creatine Kinase Is a Key Acute Phase Protein in Amphioxus Humoral Fluid Yuan-yuan Gao,† Dan-feng Zhang,† Hui Li,‡ Runzhong Liu,† Zheng-hong Zhuang,† Qi-fu Li,† San-ying Wang,† and Xuan-xian Peng*,†,‡ Center for Proteomics, Department of Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China, and State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan University, Guangzhou 510275, People’s Republic of China Received August 4, 2007
Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid companied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned. Keywords: acute phase protein • creatine kinase • amphioxus • proteomics
Acute phase proteins (APPs) contribute to the individual’s efforts to regain homeostasis as it repairs tissue damage and strives to contain and destroy potential pathogens, which plays an critical role in innate immunity of invertebrates that lack an adaptive immune system. Such a rapid, nonspecific mechanism occurs in all major taxa. Both cells and secreted molecules of innate immunity are dynamic in regard to their readiness status.3 In response to appropriate inflammatory stimulation, many of these defense-related molecules change in their concentration within a few days or less, showing that some proteins increase and the others decline as positive acute phase proteins (APPs) and negative APPs, respectively. While reasons for declines are debated, there is a general consensus that positive APPs probably all contribute to the individual’s efforts to regain homeostasis. The known positive APPs of vertebrates include clotting factors, complement components, iron-binding proteins, opsonins, anti-proteases, lectins, and others.4,5 The comparative approach has suggested the common ancestries of innate defense mechanisms in both vertebrates and invertebrates.6 Indeed, a line of evidence has indicated that the origins of APRs appear to lie in invertebrates.7 All jawed vertebrates are equipped with well-developed, basically similar adaptive immune systems in response to * To whom correspondence should be addressed at Xiamen University. Phone: (86)-592-218-7987.Fax: (86)-592-218-1015.E-mail:
[email protected]. † Xiamen University. ‡ Zhongshan University. 10.1021/pr070504x CCC: $37.00
2007 American Chemical Society
antigens. Because invertebrates and the jawless vertebrates lack T-cell receptors and major histocompatibility complex molecules that mediate adaptive immune response in vertebrates, they depend more on APR than higher vertebrates do. Santigo reported the homologue of human serum amyloid A protein (SAA) in an invertebrate deuterostome: the echinoderm Holothuria glaberrima.8 As a well-known APP, SAA increased during regeneration of the holothuroid digestive tract as compared with normal nonregenerating tissue. Gregory et al. characterized the evolution of echinoderm ferritin as an APP and further indicated that the sequestration of iron was an ancient host defense response in animals,9 which may uncover the possible evolution of an APP from invertebrates to vertebrates. Therefore, study on APPs in invertebrates may provide a novel insight into APR mechanisms in different levels of animals. Amphioxus, a cephalochordate, has been regarded as a basal lineage of chordate. It lacks free circulating blood cells in the circulation system.10 In an early study, Metchnikoff could not induce inflammation in this animal. Recently, Silva et al. have indicated that cells with morphological characteristics of phagocytes did not show in the section wound of the distal portion of amphioxus by optical and electron microscopy, but the wound was completely covered by the external cuticle of the animal at 24 h after injury. The author further indicated that amphioxus could mount an allograph rejection when the animal was tied together by suture.10 Several other reports Journal of Proteome Research 2007, 6, 4321-4329
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research articles demonstrated the presence of humoral immune-like factors in Branchiostoma belcheri tsingtauense.11,12 In addition, the acute phase response similar to that of mammalian species was observed in amphioxus.13,14 Therefore, the animal may mainly depend on fluid molecules rather than phagocytes to cope with tissue healing, infection, and other pathologies, suggesting the critical significance of humoral immunity including APR in amphioxus. Recently, our reports have indicated that proteomic methodologies for high-accuracy, high-efficiency, and highthroughput protein analyses could identify more APPs than a traditional approach.15 Therefore, the investigation on APPs of amphioxus humoral fluid by the proteomic approach will be unexceptionally interesting in this animal for an understanding of evolution characteristics of APR. The results reported here were obtained with the use of proteomic methodologies for the profile in APR-related proteins from amphioxus humoral fluid after exposure to outside injury. Interestingly, creatine kinase (CK) with several spots in line in 2-DE gels showed very strong response to the injury. The kinase was further investigated at its amount and enzyme activity in humoral fluid and tissues. Our results suggest that CK is a key molecule of APR in amphioxus.
Experimental Procedures Preparation of Amphioxus Humoral Fluid. Amphioxus [Branchiostoma belcheri (Gray)] was collected near Ocuo coast, Tong’an County, Xiamen, People’s Republic of China, and was reared in laboratory until use. The animals were fed on a diet of single-cell algae daily. The seawater was aerated continuously and kept at 23-25 °C. Five hundred animals, averaging approximately 5 cm in body length, were applied in the current study, in which 400 and 100 animals were randomly assigned into experimental and control groups, respectively. The animals in the experimental group were imposed with two caudal incisions at 0.3 cm length and 1 mm depth and then reared in laboratory. They were separately collected for humoral fluid preparation at 4, 12, 24, and 72 h, with 100 animals at every time point. Meanwhile, the 100 amphioxus of the control group were cut for humoral fluid at 0 h without caudal trauma. The preparation of humoral fluid was performed essentially as reported with a modification.11 In brief, the animals from each of the five time points in the experimental and control groups were rinsed with distilled water, then wiped thoroughly with sterilized gauze. Half of them were cut into 0.5 cm long pieces on ice and then weighted. These pooled pieces with 100 µL/g of saline solution were centrifuged at 5 000 g for 30 min at 4 °C and then supernatant was further centrifuged at 12 000 g for 30 min. The resulting supernatant was collected for CK enzyme activity analysis in 2 h or stored at -80 °C for 2-DE and 1-DE. The other half were for tissue sample collection. Prepared muscle and the others except for muscle were separately placed in Eppendorf tubes and then ice-cold saline solution was added (10 µL/mg). After grinding, these samples were centrifuged at 12 000 g for 30 min. The supernatant were collected for CK enzyme activity analysis in 2 h or stored at -80 °C for 1-DE. Protein concentration was measured by the method of Folin-phenol. Two-Dimensional Gel Electrophoresis and Image Analysis. 2-DE was performed essentially as reported previously.16 Before isoelectric focusing, the samples were dissolved in buffer (8 M urea, 4% CHAPS, 100 mM DTT, 2 M sulfocarbamide) at room temperature for 2 h. IEF was carried out with pH 3.5-9.5 carrier ampholyte for 8000 Vh. Prior to the second-dimensional gel 4322
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separation, the gel strips were equilibrated for 10 min with gentle shaking in 10 mL of SDS equilibration buffer (0.5 M TrisCl, pH 6.8, 10% glycerol, 2%SDS, 5% β-mercaptoethanol). The second-dimensional SDS-PAGE with a 12% running gel and a 5% stacking gel was carried out. After placing the gel strip on top of the second-dimensional polyacrylamide gel, electrophoresis was performed at a constant power of 100 V/gel until the bromophenol blue reached the bottom of the gel. Coomassie brilliant blue R250-stained gels were scanned with a SHARP JX-330 laser densitometer. Analysis of 2-DE and 1-DE Western blotting results was performed with the help of the software Melanie 4.0 (Swiss Institute of Bioinformatics, Geneva, Switzerland). MALDI-TOF/MS for Protein Identification. Mass spectrometric analysis was performed according to a procedure described previously.17 The protein spots of interest were excised from the CBB-stained gel, minced with a scalpel, and placed in a 1.5 mL siliconized Eppendorf tube. The digests were redissolved in 2 µL of 0.5% TFA. The solution was vortexed and centrifuged. A 1 µL portion of HCCA was mixed with the same volume of sample. Then the mixed solution was analyzed on a BRUKER autoflex MALDI-TOF (Bruker Daltonics Co.). All spectra were obtained with a positive-ion reflector. The resulting PMF, together with the pI and MW values (estimated from 2-DE gels), were searched by the program MASCOT in NCBI. ESI-MS/MS. According to the results obtained by PMF, spot 7 was randomly selected and digested as described above besides resolving in 5% formic acid, and then purified and condensed with a ZipTip instrument (Millipore, Bedford, MA). ESI-MS/MS analyses were performed on Q-Star by the Bruker Co. in Beijing. Cloning and Sequencing of CK Gene. Polyadenylated (poly A+) RNA was isolated from a whole healthy amphioxus with use of a RNeasy mRNA kit (Watson, Shanghai, China) according to the supplier’s recommendations. First strand cDNA synthesis was performed with the manufacturer’s protocol of AMV First Strand cDNA Synthesis Kit (Sangon, Shanghai, China). Primers were designed based on the CK nucleotide sequence of Branchiostoma floridae (accession AF251440) searched from the NCBI database. EcoRI and HindIII restriction sites were added to sense and antisense primers, respectively, to enable easy insertion of the CK gene into pET-32a. The sense and antisense primers of CK gene were the following: 5′-CCCGAATTCATGGCAAACTTGTGGCAG-3′ and 5′-CCCAAGCTTTTACTTGGTGCGCTTTGTC-3. Each 25 µL reaction system consisted of 2.0 µL of 25 µM sense and antisense primers (1.0 µL for each), 0.5 µL 10 mM dNTP mixture, 2.5 µL of 10 × pfu PCR buffer with MgCl2, 3 µL of first strand cDNA, 0.2 µL of 5 unit/µL of pfu DNA polymerase, and 16.8 µL of distilled water. The PCR products were fractionated on a 0.75% agarose gel in TBE (0.045 M Tris-H3BO4, 0.001 M EDTA). The gel fragments were cut out and purified by using the Geneclean II Kit (Q. BIOgene, CA). Then extracted DNA and pET-32a plasmid were separately digested with both EcoRI and HindIII at 37 °C for 2 h. Ligation products were transformed into DH5a cells. Clones were picked and cultured overnight at 37 °C. Plasmids were extracted and digested with both EcoRI and HindIII, and applied on a 0.75% agarose gel. The positive clones were sequenced and the sequence results were aligned by DNAman software. Recombinant CK Expression and Antibody Preparation. Expression of the recombinant CK was induced in E. coli BL21 cells in log phase with 1 mM IPTG and grown for an additional 3.5 h. Recombinant CK was purified by Ni-NTA affinity chro-
Cretine Kinase Is an APP in Amphioxus
matography as described in handbook. In brief, 1 mL of the 50% Ni-NAT slurry was added to 4 mL of lysate of bacteria and the solution was mixed gently by shaking for 60 min at room temperature. Then the lysate-resin mixture was loaded into an empty column, and elution fractions were collected and dialyzed by 0.01 M phosphoric buffer. After being concentrated, recombinant CK was emulsified with adjuvant. Each 0.2 mL (0.2 mL/mouse) of immunogen consisted of 0.1 mL of the protein extract and 0.1 mL of Freund’s complete adjuvant (FCA) for the first injection and Freund’s incomplete adjuvant (FIA) for the following two injections. Mice received 3 intraperitoneal injections at intervals of 2 weeks in the first and second injections and at intervals of 1 week in the third injection. We drew blood on the fifth day after the last injection and collected sera for use. SDS-PAGE and Western Blotting. The discontinuous buffer system of Laemmli with 12% resolving gels and 4% stack gels was used to resolve amphioxus humoral fluid and tissue samples. A 20 µL sample with an original volume of 0.2 µL of humoral fluid, 0.1 µL of muscle exacts, or 4 µL of other tissue exacts was heated for 5 min in boiling water and electrophoresed with a constant voltage of 100 V until the dye-front reached the bottom of the gels. The protein bands were visualized by staining with CBB. Western blotting was conducted by the method of Wu et al. with a modification.18 Humoral fluid and tissue samples from each sample were boiled for 5 min in buffer containing 10% w/v SDS, 0.3125 M Tris (pH 6.8), 0.125% bromphenol blue, 50% v/v glycerol, and 11% w/v 2-mercaptoethanol, and then separated on 12% T separating polyacrylamide gel. The separated proteins from about 30 to 60 kD in the gels were transferred to nitrocellulose membranes at 60 V constant for 2.5 h, and the other proteins in the same gels were stained with CBB for a loading control because there was no available protein marker for this animal. The papers were blocked in 5% milk and then separately incubated with a mouse antiserum to recombinant CK at a dilution of 1:1000 overnight and with goat anti-mouse horseradish peroxidase (HRP)conjugated secondary antibody at a 1:1000 dilution at 37 °C for 1 h. The papers were then washed as described previously and developed with a DAB substrate system. Phosphorylation Analysis of CK. This analysis was performed by phosphorylation of antibodies with humoral fluid and muscle samples, in which phosphatase inhibitor cocktails 1 and 2 were added during sample preparation. Three mouse monoclonal antibodies (Sigma) directed against phosphothreonine (1:5,000), phosphotyrosine (1:5,000), or phosphoserine (1: 5,000) were utilized in the analysis of Western blotting as the primary antibody. The Western blotting analysis was performed as described above. Enzyme Assay. The CK enzyme assay was performed with a commercially available kit from InTec Products, INC, Xiamen, China. Samples were obtained from the five time points of 0, 4, 12, 24, and 72 h and measured by Modular Analytics SWA of Roche according to the kit’s instructions. Statistical Analysis.The continuous data were expressed as mean ( standard deviation. The Student’s t-test was estimated by analysis of variance. Computations were performed by using SPSS 11.0. For all tests, the levels of significant difference were defined as probably less than 0.05 and 0.01.
Results Behavior and Local Wound Status Following Caudal Trauma and Characterization of Differential Proteins by Proteomic
research articles Methodologies. One hundred animals were imposed with two caudal incisions of 0.3 cm length and 1 mm depth and then reared in laboratory. Their behavior and local wound status were observed at intervals. These animals moved as normal and the caudal incision did not showed significant inflammation at 12 h, whereas they moved slowly and the wound became more severe from 24 to 72 h. There was no animal death during the experiment. 2-DE maps of amphioxus humoral fluid with 90 µg samples at 0, 4, 12, 24, and 72 h following caudal trauma were shown in Figure 1A. The profiles of the five maps were very similar, suggesting the high reproducible 2-DE in the current study. Comparison between these maps was conducted by using the automatic spot detection wizard function of Melanie 4.0 during image analysis, calculating spot intensity with relative volume divided by the total volume over the whole image, which may guarantee the quality of the quantitative data based on the same amount of sample loading. Significantly altered spots were determined in each of the four time points of the experimental group when they increased over 2-fold or decreased to less than half of the relative volume compared to the control. Nine spots with distinctly altered expression were characterized and separately named spots 1, 3, 4, 7, 8, 9, 10, 11, and 12 as shown in Figure 1A. Identification of proteins from 2-DE gels frequently involves interrogation of the genomic sequence data available for a particular species. The draft of the amphioxus genome sequences has been established in the JGI database and there are currently 886 protein sequences available in the NCBInr databases. The nine significantly differential spots were subjected to further analysis and their identities were determined by their PMF. It is interesting that spots 1, 3, 4, 7, and 8 were all identified as creatine kinase (CK). The other four spots were separately identified as ribosomal protein S10 (spot 9), CAVPtarget protein (CAVPT, spot 10), axonemal leucine-rich repeat protein (spot 11), and cathepsin (spot 12) (Table 1). Furthermore, spot 7 was randomly selected from the five CK spots and subjected to further analysis of partial sequence by ESI-MS/ MS. The obtained peptide sequence was searched against the MSDB databases, and an identical result was achieved (data not shown). Spot 1 was comparably up-regulated following caudal trauma and hit the top at 12 h, and then down-regulated until disappearance at 24 and 72 h. In contrast, spot 8 and spot 10 decreased at 12 and 24 h, and were restored at 72 h. The same goes for spots 11 and 12, but their minimum was at 4 h. Spots 3 and 4 were altered similarly and increased at 24 h. Spot 7 increased from 4 h. Spot 9 was higher at 24 h than at other points. The trend change in spots 9-12 and in the five CK spots was shown in Figure 1B and Figure 2B, respectively. However, we failed to identify more altered spots in the present study, which may be ascribed to the existence of several high abundances of proteins including CK in the humoral fluid. Thus, getting rid of the high abundance of proteins will contribute to identification of other altered proteins in this animal. Quantitative Analysis of CK Amount and Enzyme Activity in Amphioxus Humoral Fluid. We were interested in the changes of amphioxus humoral fluid CK following caudal trauma, which represented 55% of the altered spots. Interestingly, three spotssspot 2 between spot 1 and spot 3, spots 5 and 6 between spot 4, and spot 7swere in line with the five altered CK spots and also identified as CK by PMF. Therefore, a total eight CK spots in line were determined in this study. Journal of Proteome Research • Vol. 6, No. 11, 2007 4323
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Figure 1. Dynamic change in proteins during 72 h observation with the use of the 2-DE method. Samples were collected at 0, 4, 12, 24, and 72 h following caudal trauma. (A) Comparison of 2-DE maps between experimental and control groups (nine protein spots altered in APR are marked 1, 3, 4, 7, 8, 9, 10, 11, 12). Bottom right corner: SDS/PAGE map showing loading control. (B) Trend in altered spots 9-12. Volume percent represents spot intensity with relative volume divided by the total volume over the whole image, according to the Melanie 4.0 software description. The experiment was repeated at least three times. The double asterisk (**) indicates values significantly different with Student’s t-test (p < 0.01) as compared to the 0 h value. Table 1. Spots Significantly Changed Identified by PMF Searching against NCBInr Database protein description
species
score
no. of peptides matched
MW
pI
sequence coverage
AAK29780 AAK29780 AAK29780 AAK29780 AAK29780 AAO31776
creatine kinase creatine kinase creatine kinase creatine kinase creatine kinase ribosomal protein S10
68* 51* 41 86* 64* 33
10 7 5 10 8 4
42462 42462 42462 42462 42462 18145
6.09 6.09 6.09 6.09 6.09 10.07
26% 22% 17% 26% 23% 30%
10
P05548
88*
7
26775
5.89
29%
11
NP_001027749
Ciona intestinalis
63*
5
37195
4.61
30%
12
AAQ01138
CAVP-target protein (CAVPT) axonemal leucine-rich repeat protein cathepsin
Branchiostoma floridae Branchiostoma floridae Branchiostoma floridae Branchiostoma floridae Branchiostoma floridae Branchiostoma belcheri tsingtaunese Branchiostoma lanceolatum
Branchiostoma floridae
60a
6
36112
5.95
36%
spot no.
1 3 4 7 8 9
a
accession No.
Scores greater than 50 are significant (p < 0.05)
The eight CK were named CK 1-8, in which spots CK 8-2 represented a gel shift of pI19,20 (Figure 2A). Analysis was performed in every CK spot for the changes in volume at 0, 4, 4324
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12, 24, and 72 h as shown in Figure 2B. Reverse changes were determined in spot 1 at acidic pole and spot 8 at basic pole, showing an up-regulated 5.58-fold for spot 1 and a down-
Cretine Kinase Is an APP in Amphioxus
research articles
Figure 2. Comparison between eight CK spots. (A) Marked eight CK spots in different time points. (B) Trend in relative percent volume at 0, 4, 12, 24, and 72 h following caudal trauma for the eight spots and their summation. (C) Relative percent volume between the eight spots at every time point. The relative volume in parts B and C was divided by the total volume over the whole image according to the Melanie 4.0 software description. The experiment was repeated at least three times. The double asterisk (**) indicates values significantly different with Student’s t-test (p < 0.01) as compared to the 0 h value. Journal of Proteome Research • Vol. 6, No. 11, 2007 4325
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Figure 3. CK enzyme activity and amount in humoral fluid and tissues at 0, 4, 12, 24, and 72 h after injure. (A and B) 1-DE Western blotting for humoral CK (A) and its normalizing analysis (B). (C) Total CK enzyme activity in humoral fluid. (D and E) 1-DE Western blotting for muscle CK (D) and its normalizing analysis (E). (F) Total CK enzyme activity in muscle. (G and H) 1-DE Western blotting for tissue CK except for muscle (G) and its normalizing analysis (H). (I) Total CK enzyme activity in tissues except for muscle. CK enzyme activity analysis was repeated at least three times and is significantly different with Student’s t-test (p < 0.01) as compared to the 0 h value at the 0.05 level (*) and the 0.01 level (**). Western blotting analysis with mouse anti-CK as the primary antibody and SDS/PAGE analysis in the same gel as a loading control. Normalizing analysis with the use of relative percentage of CK volume by Western blotting when the volume of the control was designed as 100%.
regulated 0.38-fold for spot 8 at 12 h compared to 0 h (Figure 2B). Relative percentages of total CK in volume were 18.00%, 26.92%, 27.56%, 21.83%, and 22.54% at 0, 4, 12, 24, and 72 h, respectively, showing an increase of 1.50-, 1.53-, 1.21-, and 1.25fold at 4, 12, 24, and 72 h compared to 0 h. Further investigation of relative content between the eight spots at each time point indicated that CK 8 and 7, CK 1, 5, and 6, CK 2-4 for 0 h, CK 7 and 1, CK 6, 8, and 5, CK 2-4 for 4 h, CK 1 and 7, CK 6, CK 2-5 and 8 for 12 h, CK 7 and 6, CK 4 and 5, CK 1-3 and 8 for 24 h, CK 7 and 6, CK 8, 5 and 4, CK 1-3 for 72 h, respectively, expressed at the high, middle, and low levels (Figure 2C). Moreover, correlativity analysis was performed between CK enzyme activity and volume percent of CK 1, 3, 4, 7, and 8, showing the correlation coefficients 0.89, -0.35, -0.59, 0.80, and -0.32, respectively. Significant correlativity was achieved at p < 0.05 in only CK 1 (Pearson correlation was equal to 0.05 for CK 7 when 1-tailed was used), suggesting that CK 1 played a key role in humoral CK enzyme activity. These results suggest that the trend on down-regulation could be characterized from CK 8 to CK 2 with increasing time, whereas CK 1 was independent from CK 2 to CK 8. Furthermore, total RNA was directly used to amplify the cDNA by RT-PCR, which resulted in the amplification of a 1.140 Kb fragment. Comparison of the nucleotide sequence of the cloned sequences (DQ508694) revealed an identity with Branchiostoma floridae and Branchiostoma belcheri ranging from 95% to 96%, respectively. The deduced amino acid sequence revealed 96% identity with the sequences Accessions AAK29780 4326
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and BAE94711. The recombinant CK was purified under denaturing conditions by Ni-NTA affinity chromatography and antibodies were generated against the recombinant protein in male mice. With the use of the mouse antibodies as the primary antibodies, the altered CK was further confirmed by 1-DE Western blotting. The Western blotting analysis showed that a positive band appeared in the place corresponding to the molecular weight of CK spots in 2-DE and its reacting strength was parallel to that on 2-DE, reaching the highest point at 4 h with 127% over 0 h (Figure 3A,B). These results further demonstrated that amphioxus humoral fluid CK was regulated by caudal trauma. CK is a type of protein called an enzyme that catalyzes a biochemical reaction. Therefore, its enzyme activity should be an efficient maker for evaluating its significance in response to the injury. We separately collected amphioxus humoral fluid CK at 0, 4, 12, 24, and 72 h following caudal trauma and analyzed its enzyme activity with a commercially available kit. Our result indicated that the enzyme activity was significantly up-regulated in the humoral fluid when the animals were induced with caudal trauma. It was significantly higher at 4 and 12 h than others, showing 71, 125, 123, 82, and 77 IU/g respectively at 0, 4, 12, 24, and 72 h (Figure 3C). The alteration was highly matched between CK enzyme activity and total volume in 2-DE gel (Pearson correlation was equal to 0.94, p < 0.01), and the changing tread was also similar between CK enzyme activity and total volume identified by Western blotting, which may be related to the fact that Western blotting is not a
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truly quantitative technique especially when the HRP is used to amplify protein signal. These results suggest that CK enzyme activity showed a dynamic change following caudal trauma as APPs do. Quantitative Analysis of CK Amount and Enzyme Activity in Muscle Tissues and the Other Tissues. An elevation in the amount of CK in the human blood indicates that muscle damage has occurred, or is occurring because muscle has plenty of CK. Therefore, we further investigated the changes in the amount and enzyme activity of CK in tissues. Animal tissues were divided into two parts of muscle and the others. Both CK amount and enzyme activity were analyzed at 0, 4, 12, 24, and 72 h. Quantitative analysis by Western blotting indicated that there was no significant difference of CK amount in both muscle (Figure 3D,E) and the other tissues between the six time points (Figure 3G,H). As anticipated, CK enzyme activity was significantly higher in muscle than the others, being 902, 704, 609, 759, 750, and 796 IU/g in muscle (Figure 3F) and 11, 13, 12, 14, 13, and 13 IU/g (Figure 3I) in the other tissues respectively at 0, 4, 12, 24 and 72 h. Further enzyme assay indicated that there was no difference between the six time points of the other tissues, whereas muscle CK enzyme activity was significantly lower at 4 and 12 h than at 0 h, and at 4 h than 12 h (Figure 3F). Investigation of CK Phosphorylation Status. We further investigated whether these successive spots were caused by phosphorylation using monoclonal antibodies to phosphothreonine, phosphotyrosine, and phosphoserine. Humoral fluid and muscle samples were collected at 0, 4, 12, 24, and 72 h, and separated by SDS-PAGE. Proteins were transferred into nitrocellulose membranes, which were stained by Ponceau red S as loading control (Figure 4). Western blotting analysis indicated that no positive signal was detected at the position of CK, although positive bands reacted with the three monoclonal antibodies appeared beyond the CK position, which suggests the reliability of this detection (Figure 4).
Discussion The cephalochordate amphioxus constitutes the evolutionary link between invertebrates and vertebrates. Recently, this animal has been used as a potential model for an understanding of the evolutionary origin of an adaptive immune mechanism,21,22 suggesting the importance in characterization of its innate immunity. In the current study, five significantly altered proteins were characterized in amphioxus humoral fluid following caudal trauma with the use of proteomic methodologies. They are first reported here to be APR-related proteins in amphioxus. Out of the five altered proteins, axonemal leucine-rich repeat protein is a reference sequence in the NCBI database without function, and ribosomal protein S10 is a conceptual translation, whereas CaVPT, cathepsin, and CK have been reported in amphioxus. CaVPT, a 26-kDa endogenous target of calcium vector protein (CaVP) from amphioxus, contains three distinct regions: an N-terminal Pro-Ala-Lys-rich motif, an IQ domain, and two immunoglobulin-like folds.23 Reports have indicated that CaVP and CaVPT form a 1:1 complex by the interaction of the carboxy-terminal domain, but its physiological role is still unknown.24 Cathepsins are enzymes that have been cleaving peptide bonds of lysosomal proteins probably since lysosomes appeared in early eukaryotes.25 Phylogenetic analysis confirms the existence of two old lines of descent, the B and the L lineages of cathepsins. The amphiCB transcript with a predicted
Figure 4. Phosphorylation analysis of humoral fluid and muscle samples of amphioxus. The left panel shows the nitrocellulose membranes stained by Ponceau red S as loading control. The right panels shows Western blotting of the left gel with a mouse monoclonal antibody directed against phosphothreonine (P-Thr), phosphotyrosine (P-Tyr), and phosphoserine (P-Ser). (A) Phosphorylation analysis of amphioxus humoral fluid at 0, 4, 12, 24, and 72 h after injure. (B) Phosphorylation analysis of muscle sample at 0, 4, 12, 24, and 72 h after injure.
molecular mass of 36.5 kDa is present in all tissues of gill, muscle, testis, ovary, hepatic caecum, hind-gut, and notochord examined.26 The amphiCL has a putative signal peptide consisting of an N-terminal sequence of 15 amino acids, a propeptide containing 89 amino acid residues, and a predicted mature enzyme with 223 amino acid residues. It is expressed in the gill, testis, hepatic cecum, hind-gut, muscle, notochord, and ovary (the latter three at a low level) and up-regulated by exposure to lipopolysaccharide.27 The current study indicates the significance of the CaVPT, cathepsin, and amphiCL functioning as APPs. CK is a member of a highly conserved enzyme family called the phosphagen kinases and catalyzes the reversible transfer of phosphate from creatine phosphate to ADP yielding ATP. This enzyme is widely distributed in invertebrates and vertebrates from the sponge Tethya aurantia, the polychaete Chaetopterus variopedatus, the tunicate Ciona intestinalis, and the lancelet Branchiostoma floridae to human beings. These CKs Journal of Proteome Research • Vol. 6, No. 11, 2007 4327
research articles are strikingly similar to both invertebrate and vertebrate CKs.28 Sponges, the most primitive of all metazoans, express a dimeric CK that shows sequence homology to higher forms of CKs.29 In human beings, serum CK is documented to be a valuable marker for diagnosing tissue damage.30-31 However, information regarding to amphioxus CK function is not available although its gene was cloned for evolution analysis.28 An interesting question has arisen whether a rise of CK enzyme activity and amount in amphioxus humoral fluid results from a release by muscle damage or a feedback to caudal trauma, respectively, being a passive entry and an active defense, which may contribute to an achievement of an unknown APP function of CK. That a high total CK in the circulation could indicate damage to either the heart or other muscles has been generally accepted. Compared with our results, which indicated that CK enzyme activity was elevated at 4 h and recovered at 24 h, and showed a typically dynamic pattern of APR, accumulated data have demonstrated that the CK release resulting from tissue injury should continue for longer period so that it is often detected routinely in emergency patients including myocardial infarction and used as a recover maker.32,33 The longer the interval between the onset of symptom and blood sampling, the higher the proportion of patients with an elevation of CK-MB in acute coronary syndrome.32 Moreover, our results indicated that CK enzyme activity and amount were recovered at 24 h, but the wound was beginning to be serious at 24 h and became more serious at 72 h. A line of evidence has indicated a possible association of CK with APR in animals although the definition of CK as an APP was not suggested. Cow muscle traumas induced an APR detectable by serum haptoglobin and R(1)-acid glycoprotein correlated positively with CK.34 The plasma activity of CK was reduced in Thoroughbred horses by intramuscular injections of Freund’s complete adjuvant.35 Prior to hospital admission, elevated CK-MB was not related to myocardial infarction, myocardial ischemeia, or possible myocardial ischemeia but was associated with pneumonia, bronchitis, obstructive pulmonary disease, pulmonary embolism, chest contusion, or nonspecified pain in three patients (no detailed disease description for the three patients appeared in the paper).32 Besides, creatine kinase activity was increased in obese individuals and in neuropsychiatric lupus erythematosus (NPLE) patients.36,37 Furthermore, we investigated the source of the elevated amphioxus fluid CK in response to caudal trauma. Our results indicated that significant change in CK enzyme activity was determined in muscle, suggesting that the elevated CK in humoral fluid might be generated by muscle. As to the insignificant change in muscle CK amount between the different time points by Western blotting, it may be due to an enzyme assay being is more sensitive than Western blotting. Therefore, amphioxus CK may be a key APP in combination of the results above with the APP definition that its plasma concentration rises 25% or more following stimulation (53% and 27% elevation respectively in 2-DE and 1-DE, in the present study).38 In addition, two types of CK could be suggested based on independent CK 1 from CK 2 to 8 in amphioxus.37 Recent advances in proteomic technology have led to a number of approaches for identification of phosphoproteins, in which the gel shift of pI is a helpful clue of phosphorylation from a higher to a lower pI.19,20 The shift also appeared in CK phosphoprotein spots of chicken embryonic skeletal muscle cells when they were treated with OA or plus OAG, showing a slightly more acidic pI than the main CK subunit spot and 4328
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amounting to approximately 0.1 pH unit, as calculated from 2D-gels.39 Indeed, CK displayed interesting profiles in 2-DE maps of various samples, and was proposed to be a great example of this type of PTMs (post-translationally modified protein).37,40 These results are in agreement with our findings on CK 8-CK 2. Thus we may suggest the enhanced phosphorylated degree from CK 2 to CK 8 following caudal trauma. However, we did not observe CK phosphorylation on threonine, tyrosin, and serine residues in both muscle and humoral fluid of amphioxus using monoclonal antibodies to phosphorylation. A recent report has revealed that six phosphorylated residues were phosphothreonine (P-Thr) is in the vicinity of the active site of chicken MM-CK,41 four of them exist in amphioxus CK (Thr-282, -289-322, and -327). In the present study, the failure to detect CK phosphorylation by phosphorylation-specific antibodies may result from steric hindrance of the recognition site, especially with P-Thr and P-Ser antibodies.42 Also the phosphorylated residues of amphioxus CK may be beyond the three amino acids of threonine, tyrosin, and serine. On the other hand, the eight CKs showed totally different dynamic patterns following the time intervals of injury, which may suggest that these CKs with different PTMs play different roles in response to the caudal trauma.
Acknowledgment. This work was sponsored by grants from the National Basic Research Program of China (2006CB101807), NSFC project 30530610, and the open project of the State Key Laboratory of Biocontrol (2006-06). References (1) Gerwick, L.; Corley-Smith, G. E.; Nakao, M.; Watson, J.; Bayne, C. J. Intracranial injections induce local transcription of a gene encoding precerebellin-like protein. Fish Physiol. Biochem. 2005, 31, 363-372. (2) Lin, B.; Chen, S. W.; Cao, Z.; Lin, Y. Q.; Mo, D. Z.; Zhang, H. B.; Gu, J. D.; Dong, M. L.; Liu, Z. H.; Xu, A. L. Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: Striking similarities and obvious differences with mammals. Mol. Immunol. 2007, 44, 295-301. (3) Beck, G.; Habicht, G. S.; Cooper, E. L.; Marchalonis, J. J.; Primordial immunity: foundations of the vertebrate immune system. Ann. N.Y. Acad. Sci. 1994, 712-736. (4) Steel, D. M.; Whitehead, A. S. The major acute phase reactants: C-reactive protein, serum amyloid P and serum amyloid A protein. Immunol. Today 1994, 15, 81-88. (5) Bayne, C. J.; Gerwick, L. The acute phase response and innate immunity of fish. Dev. Comp. Immunol. 2001, 25, 725-743. (6) Hoffmann, J. A.; Kafatos, F. C.; Janeway, C. A., Jr.; Ezekowitz, R. A. Phylogenetic Perspectives in Innate Immunity. Science 1999, 284 1313-1318. (7) Armstrong, P. B.; Quigley, J. P. Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system. Dev. Comp. Immunol. 1999, 23, 375-390. (8) Santiago, P.; Roig-Lo´pez, J. L.; Santiago, C.; Garcı´a-Arrara´, J. E. Serum amyloid A protein in an echinoderm:its primary structure and expression during intestinal regeneration in the sea cucumber Holothuria glaberrima. Exp. Zool. (Mol. Dev. Evol.) 2000, 288, 335-344. (9) Beck, G.; Ellis, T. W.; Habicht, G. S.; Schluter, S. F.; Marchalonis, J. J. Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule. Dev. Comp. Immunol. 2002, 26 (1), 11-26. (10) Silva, J. R. M. C.; Mendes, E. G.; Mariano, M. Wound repair in the amphioxus (Brachiostoma platae), an animal deprived of inflammatory phagocytes. J. Invert. Path. 1995, 65, 147-151. (11) Zhang, S. C.; Wang, C. F.; Wang, Y. J.; Wei, R.; Jiang, G. H.; Ju, H. Presence and characterization of complement-like activity in the amphioxus branchiostoma belcheri tsingtauense. Zool. Sci. 2003, 20, 1207-1214. (12) Sato, A.; Mayer, W. E.; Klein. J. A molecule bearing an immunoglobulin-like V region of the CTX subfamily in amphioxus. Immunogenetics 2003, 55, 423-427.
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