Molecular characterization of pig LEG1a protein

doi:10.3785/j.issn.1008-9209.2021.04.081

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

Issue (2): 261-268 DOI: 10.3785/j.issn.1008-9209.2021.04.081

Animal sciences & veterinary medicines

Molecular characterization of pig LEG1a protein

Minghao SUN(

),Yuqi HUANG,Yanna DANG,Jin HE(

)

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

Download:

HTML

HTML ( )

PDF(1058KB)
Export: BibTeX | EndNote (RIS)

Abstract

Liver-enriched gene 1 (LEG1) is a newly identified gene that plays an important role in the liver development of zebrafish and the innate immunity of platypus. However, little is known about LEG1 in eutherians such as mice and humans. In this study, we explored the molecular characteristics of the LEG1 protein in a pig model (pLEG1a). A rabbit polyclonal antibody against pLEG1a was produced and validated. Then, we detected the pLEG1a protein in both the salivary gland and lung. Moreover, pLEG1a could be detected in the saliva, suggesting its secretory nature. Ectopic expression of pLEG1a in HEK293T cells further confirmed that pLEG1a was a secretory protein. In addition, glycosylation tests showed the existence of glycosylated bands which could be enzymatically removed, indicating that the secreted pLEG1a was N-glycosylated. In conclusion, the pLEG1a protein is similar to the LEG1 proteins of other species, especially eutherian LEG1s, in terms of expression profile, glycosylation, and secretion ability, suggesting that pLEG1a is a good model to further study the function of eutherian LEG1s.

Key words： pig LEG1a protein secretory protein glycosylation

Published: 25 April 2022

CLC:

S 828

Fund: National Nature Science Foundation of China(31802029);the Special Funds of the National Nature Science Foundation of China(81941003);the Fundamental Research Funds for the Central Universities of China(2020XZZX002-20)

Corresponding Authors: Jin HE E-mail: mhsun29@zju.edu.cn;hejin@zju.edu.cn

About author: SUN Minghao (https://orcid.org/0000-0002-8317-5847), E-mail: mhsun29@zju.edu.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Minghao SUN
	Yuqi HUANG
	Yanna DANG
	Jin HE

Cite this article:

Minghao SUN, Yuqi HUANG, Yanna DANG, Jin HE. Molecular characterization of pig LEG1a protein. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(2): 261-268.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.04.081 OR https://www.zjujournals.com/agr/Y2022/V48/I2/261

猪的肝富集基因1a蛋白的分子鉴定(英文)

新发现的肝富集基因1（live enriched-gene 1, LEG1）在斑马鱼肝脏发育及鸭嘴兽的先天性免疫应答过程中发挥着重要的调控作用，但是LEG1在真兽亚纲物种（如小鼠和人）中的功能鲜有报道。本研究以猪为模型，对猪的肝富集基因1a蛋白（pLEG1a）进行了分子鉴定。实验首先制备并验证了pLEG1a蛋白的兔源多克隆抗体，并验证了pLEG1a在猪唾液腺和肺部的表达。同时，在唾液中检测到了pLEG1a蛋白的信号，表明pLEG1a蛋白可能是一种分泌蛋白。此外，当在HEK293T细胞中异位表达pLEG1a时，可在细胞培养液中检测到pLEG1a蛋白，进一步证明了pLEG1a是一种分泌型蛋白。另外，糖基化实验显示，唾液及HEK293T细胞中的pLEG1a存在糖基化条带，且这一条带可以通过糖基化酶去除，表明pLEG1a是一种N-糖基化蛋白。综上所述，本研究探究了pLEG1a蛋白与其他物种（尤其是真兽亚纲动物）LEG1蛋白在表达谱、糖基化和分泌能力方面的相似性，并且提出pLEG1a可能是研究真兽亚纲物种LEG1基因的良好模型。

关键词： 猪, 肝富集基因1a蛋白, 分泌蛋白, 糖基化

Fig. 1 Western blotting analysis of the pLEG1 proteinA. Detection of pLEG1a by anti-pLEG1a (No. 1869) and anti-hLEG1a (No. 897) antibodies and antiserums (M: Marker; 1: Heart; 2: Salivary gland; 3: pCMV-pleg1a-C-FLAG transiently transfected HEK293T cells. Red arrow corresponds to an ≈38 kDa band); B. Detection of pLEG1a and pLEG1b by anti-pLEG1a (No. 1869) antibody [M: Marker; nc: Non-specific control; 1a: pCAG-pleg1a-3

×

FLAG-transfected cell extract; 1b: pCAG-pleg1b-3

×

HA-transfected cell extract. Left panel: Extracts from cells transfected with pCAG-pleg1a-3

×

FLAG and pCAG-pleg1b-3

×

HA were blotted with anti-pLEG1a (No. 1869) antibody, and the red arrowhead indicates the detected band. Right panel: The same samples were blotted with an anti-FLAG antibody, resulting in only one band detected from the pCAG-pleg1a-3

×

FLAG cell extract (red arrowhead)].

Fig. 2 Expression of the pLEG1a proteinA. Detection of pLEG1a in various tissues using a rabbit anti-pLEG1a antibody (No. 1869) (SG: Salivary gland; SM: Skeletal muscle; LI: Large intestine; SI: Small intestine; Br: Brain; Ki: Kidney; Lu: Lung; Sp: Spleen; Li: Liver; He: Heart); B. Detection of pLEG1a in concentrated saliva by Western blotting [Saliva 1, 2, and 3 indicate the saliva collected from three different pigs (Western blotting analysis for saliva 3 was performed in a separate blot). The red arrows show the nonglycosylated pLEG1a, while the blue arrow denotes the glycosylated pLEG1a, which is slightly lower than the nonspecific 55 kDa band in the tissues].

Fig. 3 Glycosylation test of pLEG1aA. Glycosylation prediction of pLEG1a, pLEG1b, and pLEG1c (N-glycosylation sites are highlighted in red, while O-glycosylation sites are highlighted in blue); B. Deglycosylation test of pLEG1a in the heart and salivary gland extracts (1: No treatment; 2: PNGase F treatment; 3: O-glycosidase/neuraminidase treatment); C. Deglycosylation test of saliva pLEG1a using PNGase F or O-glycosidase/neuraminidase [The same polyvinylidene difluoride membrane was exposed for a short time (lower panel) and a long time (upper panel)]; D. Western blotting analysis of transiently transfected HEK293T cells expressing either pCAG-pleg1a-3

×

FLAG or pCAG-pleg1b-3

×

HA using anti-epitope antibodies (CCM: Cell culture medium; M: Marker; nc: Non-specific control; 1a: pCAG-pleg1a-3×FLAG-transfected cell extract; 1b: pCAG-pleg1b-3×HA-transfected cell extract).


[1]	CHANG C Q, HU M J, ZHU Z H, et al. Liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish[J]. PLoS ONE, 2011, 6(8): e22910. DOI:10.1371/journal.pone.0022910 doi: 10.1371/journal.pone.0022910

[2]	HU M J, BAI Y, ZHANG C X, et al. Liver-enriched gene 1, a glycosylated secretory protein, binds to FGFR and mediates an anti-stress pathway to protect liver development in zebrafish[J]. PLoS Genetics, 2016, 12(2): e1005881. DOI:10.1371/journal.pgen.1005881 doi: 10.1371/journal.pgen.1005881

[3]	ENJAPOORI A K, GRANT T R, NICOL S C, et al. Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection[J]. Genome Biology and Evolution, 2014, 6(10): 2754-2773. DOI:10.1093/gbe/evu209 doi: 10.1093/gbe/evu209

[4]	DANG Y, WANG J Y, LIU C, et al. Evolutionary and molecular characterization of liver-enriched gene 1 [J]. Scientific Reports, 2020, 10(1): 4262. DOI:10.1038/s41598-020-61208-7 doi: 10.1038/s41598-020-61208-7

[5]	ZHANG Z, WANG Z, DANG Y, et al. Transcriptomic prediction of pig liver-enriched gene 1 functions in a liver cell line[J]. Genes, 2020, 11(4): 412. DOI:10.3390/genes11040412 doi: 10.3390/genes11040412

[6]	PINI T, LEAHY T, SOLEILHAVOUP C, et al. Proteomic investigation of ram spermatozoa and the proteins conferred by seminal plasma[J]. Journal of Proteome Research, 2016, 15(10): 3700-3711. DOI:10.1021/acs.jproteome.6b00530 doi: 10.1021/acs.jproteome.6b00530

[7]	RAMACHANDRAN P, BOONTHEUNG P, XIE Y M, et al. Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry[J]. Journal of Proteome Research, 2006, 5(6): 1493-1503. DOI:10.1021/pr050492k doi: 10.1021/pr050492k

[8]	BENSADOUN A, WEINSTEIN D. Assay of proteins in the presence of interfering materials[J]. Analytical Biochemistry, 1976, 70(1): 241-250. DOI:10.1016/s0003-2697(76)80064-4 doi: 10.1016/s0003-2697(76)80064-4

[9]	STEENTOFT C, VAKHRUSHEV S Y, JOSHI H J, et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology[J]. The EMBO Journal, 2013, 32(10): 1478-1488. DOI:10.1038/emboj.2013.79 doi: 10.1038/emboj.2013.79

[10]	NYNCA J, ARNOLD G J, FROHLICH T, et al. Proteomic identification of rainbow trout seminal plasma proteins[J]. Proteomics, 2014, 14(1): 133-140. DOI:10.1002/pmic.201300267 doi: 10.1002/pmic.201300267

[11]	RAMACHANDRAN P, BOONTHEUNG P, PANG E, et al. Comparison of N-linked glycoproteins in human whole saliva, parotid, submandibular, and sublingual glandular secretions identified using hydrazide chemistry and mass spectrometry[J]. Clinical Proteomics, 2008, 4(3/4): 80-104. DOI:10.1007/s12014-008-9005-0 doi: 10.1007/s12014-008-9005-0

[12]	ZHU Z H, HU M J, CHANG C Q, et al. Analysis of expression pattern of zebrafish leg1 homologus gene mu-leg1 in mouse[J]. Hereditas, 2012, 34(9): 1174-1180. DOI:10.1007/s11783-011-0280-z doi: 10.1007/s11783-011-0280-z

[13]	KARN R C, CHUANG A G, LAUKAITIS C M. Shared and unique proteins in human, mouse and rat saliva proteomes: footprints of functional adaptation[J]. Proteomes, 2013, 1(3): 275-289. DOI:10.3390/proteomes1030275 doi: 10.3390/proteomes1030275

[14]	HELENIUS A, AEBI M. Intracellular functions of N-linked glycans[J]. Science, 2001, 291(5512): 2364-2369. DOI:10.1126/science.291.5512.2364 doi: 10.1126/science.291.5512.2364

[15]	HAMILTON J A, BENSON M D. Transthyretin: a review from a structural perspective[J]. Cellular and Molecular Life Sciences, 2001, 58(10): 1491-1521. DOI:10.1007/PL00000791 doi: 10.1007/PL00000791

[16]	GUAN J L, MACHAMER C E, ROSE J K. Glycosylation allows cell-surface transport of an anchored secretory protein[J]. Cell, 1985, 42(2): 489-496. DOI:10.1016/0092-8674(85)90106-0 doi: 10.1016/0092-8674(85)90106-0

[17]	HENKEL J R, GIBSON G A, POLAND P A, et al. Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized Madin-Darby canine kidney cells[J]. Journal of Cell Biology, 2000, 148(3): 495-504. DOI:10.1083/jcb.148.3.495 doi: 10.1083/jcb.148.3.495

[18]	SATO T, SAKO Y, SHO M, et al. STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein[J]. Molecular Cell, 2012, 47(1): 99-110. DOI:10.1016/j.molcel.2012.04.015 doi: 10.1016/j.molcel.2012.04.015

[1]	Haitian CHEN,Xuejun ZHOU,Junjing SHA,Xiaoli LI,Jin WANG,Yong HE. Changes of physiological and biochemical indexes of tea plant leaves under lead aerosol stress and their rapid spectral detection[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(1): 117-128.

[2]	Jinzhu MENG,Zhenyang WU,Qingming AN,Yuanyuan ZHAO. Screening of genes related to intramuscular fat deposition in Qianbei black pigs[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(6): 797-804.

[3]	Chunmiao JI,Bin WANG,Pan QIN,Yaowei HUANG. Analysis of the characteristics of porcine antibody repertoire by high-throughput sequencing method[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(5): 660-666.

[4]	Zhen WEI,Ke HE,Shenghui HONG,Diwen LIU. Polymorphism and differential expression of MHCclassⅠgenes between different strains of guinea pig[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(4): 534-542.

[5]	Xinxin YOU,Sheng WANG,Linna DU. Characteristics of an endogenous compound microbial inoculant and its immobilization effect on wastewater treatment from pig feedlots[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(1): 98-106.

[6]	Qianqian YANG,Jianguo CAI,An WANG,Yanwei LIU. *Growth and aluminum absorption responses of Hydrangea macrophylla* to combined treatments of acid rain and aluminum**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(6): 718-726.

[7]	Wenxue HAN,Hao JIANG,Jian BIAN,Jinhu YUN,Yanyan SUN,Wangxiang ZHANG,Ye PENG. Leaf color change and its correlation with pigment content in 10 ornamental crabapple varieties in spring[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(5): 562-570.

[8]	Yueling ZHAO,Jian DING,Jia HE,Dedong KONG,Ping XU,Yuefei WANG. Protective effect of epigallocatechin gallate on inflammatory bowel disease induced by dextran sulfate sodium in mice[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 626-634.

[9]	Fuyin HOU,Yingjiang CHEN,Zhiqing YANG,Chongfu JIN,Kai SHI,Changkuan CHEN,Gongneng FENG,Hongshan LI. *Effects of digested pig slurry application on agronomic trait, yield and forage quality of indica* rice**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 325-331.

[10]	Yatao HE,Dandan GAO,Senning GAN,Ting SUN,Kuizheng CAI,Junlin LIU. *Isolation and identification of a red pigment producer endophytic fungus Monascus sanguineus* from Rehmannia glutinosa Libosch**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 1-7.

[11]	Li ZHANG,Weilun NONG,Jianxiong LU,Guohua ZHANG,Lixia LIU. *Single nucleotide polymorphism screening and bioinformatics analysis of the main family genes of FABPs* in Bamei pig**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 109-118.

[12]	LIU Tian, CHEN Xiaoyu, ZHU Zhiwei, YU Fuxian, HUANG Jing, JIA Ruoxin, SHI Fangxiong, PAN Jianzhi. Screening of follicle-stimulating hormone dose-dependent expression genes[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(2): 162-171.

[13]	FAN Qiwen, CHEN Lingli, ZHAO Li, SHI Min, YAN Xianghua. Effect of leucine on endoplasmic reticulum stress induced by early weaning in the liver of piglets[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(2): 172-180.

[14]	MA Lianxiang, HOU Chuanchuan, HE Junna, QIU Jialing, LU Xintao, GUO Yang, LIU Bing, LIN Gang, XUE Yan, YU Dongyou. Effect of compound organic trace minerals on growth performance, serum indexes and micromineral excretion in fattening pigs[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(2): 181-189.

[15]	ZHENG Youxiu, WANG Chao, YAO Jingyi, LU Jianjun. *Effects of Clostridium butyricum* on serum biochemical indices, antioxidant function and intestinal microbiota in weaned piglets** [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(2): 190-198.

Viewed

Full text

Abstract

Cited

Shared

Discussed