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Definition

Intermedin (IMD) is a novel member of the calcitonin gene-related peptide (CGRP) family which also includes adrenomedullin (AM).

Discovery

In early 2004, a novel peptide was independently identified by two separate groups. Roh and colleagues initially identified human IMD from an expressed sequence tag and used phylogenetic profiling to identify the peptide in other mammals and nonvertebrates. Immunohistochemical analysis of peptide expression revealed that high levels of the peptide and/or preprohormone were present in cells within the intermediate lobe of the pituitary; hence, the group called the peptide intermedin1. At the same time, Takei et al. were examining a rat cDNA library for potential mammalian homologs of the five identified puffer fish adrenomedullin (AM) family members. They identified a 148-amino acid preprohormone that was processed into a 47-amino acid, mature peptide with 33% sequence homology to AM and 71% homology to puffer fish AM2 and thus referred to the peptide as mammalian AM22.

Structural Characteristics

The human IMD gene is believed to encode a prepropeptide of 148 amino acids with a signal peptide for secretion at the N terminus, which may generate a 47-amino acid mature peptide (IMD1–47) i.e. IMDL and a shorter 40-amino acid one (IMD8–47) i.e.  IMDS by proteolytic cleavage at the N-terminal proximate basic residues followed by an amidated C terminus. Intermedins has a stretch of 47 residues at the C terminus is flanked by dibasic proteolytic cleavage sites at the N terminus and an a-amidation donor residue at the C terminus. The putative mature region of intermedin shares 28% sequence identity with ADM and 20% with CGRP. Importantly, Intermedins adopts an N-terminal disulfide- bonded loop leading into an a-helix, followed by a disordered structure that is shared by all calcitonin/CGRP family peptides. Analysis of orthologous intermedins indicate that the positions of N-terminal dibasic cleavage sites vary by a few amino acids among different species, whereas an arginine residue seven amino acids downstream of the dibasic cleavage motif of human intermedin is conserved in all species, suggesting that the mature intermedin from human and other species could be a 40-amino acid peptide3.

Mode of action

Intermedin activates the cAMP-dependent Pathway via the CGRP Receptor— A study shows that treatment of human neuroblastoma SK-N-MC cells and rat L6 skeletal myoblast cells, known to express different levels of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMPs), with synthetic intermedin peptides resulted in increased cAMP production, whereas  treatment with the  truncated amidated intermedin fragment (intermedin (IMD)- (17–47)), or a 31-amino acid peptide from the preproregion of human intermedin (prointermedin-(55–85)) had no effect in either cell line. This implied that a-amidation and residues 8–16 of intermedin are important for intermedin bioactivity. Further it was also shown that the stimulatory effect of intermedin was suppressed by cotreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP-(8–37)) in L6 cells3.

Functions

Intermedin/Adrenomedullin-2 elevate blood pressure and inhibit food and water intake - Intermedin is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. It has been reported that administration of IMD into the lateral cerebroventricle (i.c.v.) of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by i.c.v. administration of phentolamine indicating that IMD activates the sympathetic nervous system. I.c.v administration of IMD also inhibited food and water intake. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. A study indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both the AM and CGRP receptors4.

Expression of the Counter-Regulatory Peptide Intermedin is Augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes - A study was done using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY), to examine the presence of myocardial oxidative stress, concentric hypertrophy expression of IMD, AM and receptor components. It was found that in left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%) and IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered and abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. This implied that robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension5.

References

1.     Roh J, Chang CL, Bhalla A, Klein C, and Hsu SYT (2004). Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complex. J. Biol. Chem., 279, 7264–7274.

2.     Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, and Miyano S (2004). Novel fish-derived adrenomedullin in mammals: structure and possible function. Peptides, 25, 1643- 1656.

3.     Jaesook Roh, Chia Lin Chang, Alka Bhalla, Cynthia Klein, and Sheau Yu Teddy Hsu (2004). Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family Peptide Acting through the Calcitonin Receptor-liken Receptor/Receptor Activity-modifying Protein Receptor Complexes. J. Bio. Chem., 279 (20), 7264–7274.

4.     Taylor MM, Bagley SL, Samson WK (2004). Intermedin/Adrenomedullin-2 acts within the central nervous system to elevate blood pressure and inhibit food and water intake. Am. J Physiol. Regul. Integr. Comp. Physiol., 288, R919-R927.

5.     David Bell, Y Zhao, Francis PG. McCoy, Adrian Devine and Barbara J. McDermott (2008). Expression of the Counter-Regulatory Peptide Intermedin is augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes. Cell. Physiol. Biochem., 21, 409-420.

多肽H2N-Asp-Glu-Gly-Pro-Tyr-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-COOH的合成步骤：

1、合成CTC树脂：称取1.48g CTC Resin（如初始取代度约为0.87mmol/g）和1.55mmol Fmoc-Asp(OtBu)-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3.86mmol 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-Asp(OtBu)-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-Asp(OtBu)-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3.86mmol Fmoc-Lys(Boc)-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7.73mmol DIPEA，3.67mmol 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-Lys(Boc)-Asp(OtBu)-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-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Glu(OtBu)-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

H2N-Glu(OtBu)-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-CTC Resin

Fmoc-Asp(OtBu)-Glu(OtBu)-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-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-Asp(OtBu)-Glu(OtBu)-Gly-Pro-Tyr(tBu)-Lys(Boc)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Ser(tBu)-Pro-Pro-Lys(Boc)-Asp(OtBu)-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-Asp-Glu-Gly-Pro-Tyr-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-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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