400-998-5282
专注多肽 服务科研
400-998-5282
专注多肽 服务科研
P16是人C3d的一个由16个氨基酸组成的肽序列，它根据细胞分化的阶段，在体外和体内调节pp100和pp105的酪氨酸磷酸化，以及人B淋巴细胞的体外增殖。
编号：142320
CAS号：99027-06-2
单字母：H2N-KNRWEDPGKQLYNVEA-OH
| 参考文献（References）: | R.Frade et al., Biochem. Biophys. Res. Commun., 188, 833 (1992) |
P16是人C3d的一个由16个氨基酸组成的肽序列，它根据细胞分化的阶段，在体外和体内调节pp100和pp105的酪氨酸磷酸化，以及人B淋巴细胞的体外增殖。
P16, a 16-amino acid peptide sequence of human C3d, regulated in vitro and in vivo tyrosine phosphorylations of the pp100 and pp105 and in vitro proliferation of human B lymphocytes, depending on the stage of cell differentiation.
Definition
C3a is an anaphylotoxin and a cleavage product of the major complement protein C31. C3d, important for enhancing B cell responses is produced by cleavage of C3b which is also another product of C31.
Discovery
Complement system was first discovered in the 1880’s as a heat liable bactericidal activity component in fresh serum2. Later several components of the complement proteins were identified by protein purification techniques3. C3 and C3d were first isolated from the waste of industrially produced albumin in 19604.
Classification
C3a, C4a and C5a serum proteins of the complement system are all anaphylotoxins5. C3d belongs to the class of antigen binding proteins6.
Structural Characteristics
C3a is a 77 amino acid peptide with a random coiled C-terminus and a well defined helical N terminus7,8. C3d consists of an acidic residue lined electronegative depression that is away from the antigen binding site. This depression can bind the receptor which is electropositive in nature9.
Mode of action
C3a exerts its functions by binding to the G protein coupled receptor-C3a receptor. Upon binding of C3a to the receptor cAMP levels are triggered which in turn activates downstream signaling pathways10.
A receptor for C3d fragment of C3dg exists on B lymphocytes, B lymphoblastoid cell lines, and follicular dendritic cells. This receptor, termed complement receptor (CR2), is important in B lymphocyte proliferation and differentiation. CR2 interacts with each of the C3 fragments containing residues 1209 to 1236 of C3d very strongly with C3d11,12.
Functions
C3a plays an important role in chemotaxis13. It increases the production of cytokines such as IL-6 thereby influencing the immune response14. It is also important for liver regeneration after tissue injury15.
C3d acts as an adjuvant by binding the antigen to the B cell through the CR2 receptor hence enhancing B cell humoral responses16.
References
1. Hugli TE (1986). Biochemistry and biology of anaphylatoxins. Complement, 3 (3), 111–27.
2. Buchner H (1889). Uber die bakterientodtende, Wirkunk des zellenfrien Blutserums. Zentralbl Bakteriol, 5, 1-11.
3. Muller-EHJ (1968). Chemistry and reaction mechanisms of complement. Adv Immunol., 8,1-80.
4. Matveevskaya NS, Alyoshkin VA and Rozina MN (1995). Isolation of the C3 complement component and its C3d subunit from IY-1 fraction of Cohn's fractionation of human plasma. J of chromatography B: Biosciences and applications, 664, 1, 261-66.
5. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Köhl J (2009). The role of the anaphylatoxins in health and disease. Molecular Immunology [EPub ahead of print]
6. Bergmann-Leitner ES, Leitner WW, Tsokos GC (2006). Complement 3d: from molecular adjuvant to target of immune escape mechanisms. Clin Immunol., 121(2), 177-85.
7. Maarten HL De Brunn and Georg HF (1985). Human complement component C3: cDNA coding sequence and derived primary structure (DNA sequence analysis/proteolytic cleavage site/signal peptide/precursor protein/family of plasma proteins. Proc. Natl. Acad. Sci. USA, 82, 708-712.
8. Nettesheim DG, Edalji RP, Mollison KW, Greer J, and Zuiderweg ER (1988). Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations. Proc Natl Acad Sci USA, 85(14), 5036–5040.
9. Liliana C and David EI (2000). Structure-Guided Identification of C3d Residues Essential for Its Binding to Complement Receptor 2 (CD21)1. J Immunol., 165, 3839-3848.
10. Alison AH, Bao L, Christy AN, Craig L, Elliot I, Yuko F, Norma PG & Craig G (2000). A role for the C3a anaphylatoxin receptor in the effector phase of asthma. Nature 406, 998-1001.
11. Köhl J (2006). Self, non-self, and danger: a complementary view. Adv Exp Med Biol., 586, 71-94.
12. Paul MK, Daniel SR, Si-Han H, Julie McM, William M and Anne S De G (2008). Novel function of complement C3d as an autologous helper T-cell target. Immunology and Cell Biology, 86, 221–225.
13. Britt S, Daniel B, Henry S, So¨ren G, Claudia R, Jo¨rg K, Wilfried B, and Andreas K (1999). Modulation of C3a Activity: Internalization of the Human C3a Receptor and its Inhibition by C5a1. J Immnunol., 162, 7409–7416.
14. Fischer WH, Jagels MA, Hugli TE (1999). Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). J Immunol.,1,162(1), 453-9.
15. Christopher WS, Maciej M, Dimitrios M, Ruxandra T, Lynn AS, Linda EG and John DL (2003). The Proinflammatory Mediators C3a and C5a Are Essential for Liver Regeneration. J Exp Med., 198, 6, 913-923.
16. Karen MH, Franklin RT, Julie AO, Jonathan CP, John HW, David RK, Joseph FB, Ted MR and Thomas FT (2004). Cutting Edge: C3d Functions as a Molecular Adjuvant in the Absence of CD21/35 Expression. J Immunol., 172 (10), 5833.
多肽H2N-Lys-Asn-Arg-Trp-Glu-Asp-Pro-Gly-Lys-Gln-Leu-Tyr-Asn-Val-Glu-Ala-COOH的合成步骤：
1、合成CTC树脂：称取1.21g CTC Resin（如初始取代度约为0.63mmol/g）和0.91mmol Fmoc-Ala-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入2.29mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到 Fmoc-Ala-CTC Resin。结构图如下：
2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Ala-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：
3、缩合：取2.29mmol Fmoc-Glu(OtBu)-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入4.57mmol DIPEA，2.17mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Glu(OtBu)-Ala-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：
4、依次循环步骤二、步骤三，依次得到
H2N-Glu(OtBu)-Ala-CTC Resin
Fmoc-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Asn(Trt)-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
H2N-Asn(Trt)-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
Fmoc-Lys(Boc)-Asn(Trt)-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin
以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。
最后再经过步骤二得到 H2N-Lys(Boc)-Asn(Trt)-Arg(Pbf)-Trp(Boc)-Glu(OtBu)-Asp(OtBu)-Pro-Gly-Lys(Boc)-Gln(Trt)-Leu-Tyr(tBu)-Asn(Trt)-Val-Glu(OtBu)-Ala-CTC Resin，结构如下：
5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Lys-Asn-Arg-Trp-Glu-Asp-Pro-Gly-Lys-Gln-Leu-Tyr-Asn-Val-Glu-Ala-COOH。结构图见产品结构图。
切割液选择：1）TFA：H2O=95%：5%
2）TFA：H2O：TIS=95%：2.5%：2.5%
3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%
（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）
6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。
7、最后总结：
杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。
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