Antibody upstream sequence diversity and its biological implications revealed by repertoire sequencing

Yan Zhu; Xiujia Yang; Cuiyu Ma; Haipei Tang; Qilong Wang; Junjie Guan; Wenxi Xie; Sen Chen; Yuan Chen; Minhui Wang; Chunhong Lan; Deqiang Sun; Lai Wei; Caijun Sun; Xueqing Yu; Zhenhai Zhang

doi:10.1016/j.jgg.2021.06.016

Volume 48 Issue 10

Oct. 2021

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2021 > 48(10): 936-945

PDF( 2378 KB)

Antibody upstream sequence diversity and its biological implications revealed by repertoire sequencing

doi: 10.1016/j.jgg.2021.06.016

Yan Zhu^a,b,c,d,e,
Xiujia Yang^a,b,c,d,e,
Cuiyu Ma^a,b,
Haipei Tang^c,
Qilong Wang^c,
Junjie Guan^a,b,
Wenxi Xie^a,b,
Sen Chen^a,b,
Yuan Chen^c,
Minhui Wang^a,f,g,
Chunhong Lan^a,c,
Deqiang Sun^h,
Lai Weiⁱ,
Caijun Sun^j,
Xueqing Yu^d,k,
Zhenhai Zhang^a,b,c,d,e

a. State Key Laboratory of Organ Failure Research, National Clinical Research Center for Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;
b. Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China;
c. Center for Precision Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China;
d. Guangdong-Hong Kong Joint Laboratory on Immunological and Genetic Kidney Diseases, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China;
e. Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Southern Medical University, Guangzhou 510515, China;
f. Department of Nephrology, Hainan Affiliated Hospital of Hainan Medical College, Haikou 570311, China;
g. Department of Nephrology, Hainan General Hospital, Haikou 570311, China;
h. Department of Center Laboratory, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, China;
i. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China;
j. School of Public Health, Sun Yat-sen University, Shenzhen 510006, China;
k. Division of Nephrology, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China

Funds:

This study was supported by the National Natural Science Foundation of China (NSFC) (31771479 to Z.Z.), NSFC Projects of International Cooperation and Exchanges of NSFC (61661146004 to Z.Z.), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S131 to Z.Z.), and Guangdong-Hong Kong-Macao-Joint Labs Program from Guangdong Science and Technology (2019B121205005 to X.Y.).

Received Date: 2021-04-30
Accepted Date: 2021-06-16
Rev Recd Date: 2021-06-10
Publish Date: 2021-07-16

Abstract

Abstract

The sequence upstream of the antibody variable region (antibody upstream sequence [AUS]) consists of a 5' untranslated region (5' UTR) and a preceding leader region. The sequence variations in AUS affect antibody engineering and PCR based antibody quantification and may also be implicated in mRNA transcription and translation. However, the diversity of AUSs remains elusive. Using 5' rapid amplification of cDNA ends and high-throughput antibody repertoire sequencing technique, we acquired full-length AUSs for human, rhesus macaque, cynomolgus macaque, mouse, and rat. We designed a bioinformatics pipeline and identified 3307 unique AUSs, corresponding to 3026 and 1457 unique sequences for 5' UTR and leader region, respectively. Comparative analysis indicated that 928 (63.69%) leader sequences are novel relative to those recorded in the international ImMunoGeneTics information system. Evolutionarily, leader sequences are more conserved than 5' UTR and seem to coevolve with their downstream V genes. Besides, single-nucleotide polymorphisms are position dependent for leader regions and may contribute to the functional reversal of the downstream V genes. Finally, the AUGs in AUSs were found to have little impact on gene expression. Taken together, our findings can facilitate primer design for capturing antibodies efficiently and provide a valuable resource for antibody engineering and molecule-level antibody studies.
- Antibody upstream sequences,
- 5′ UTR,
- Leader sequences,
- Antibody repertoire sequencing,
- Antibody repertoire

These authors contributed equally to this work.

FullText(HTML)

References(42)

References

Boyd, S.D., Marshall, E.L., Merker, J.D., Maniar, J.M., Zhang, L.N., Sahaf, B., Jones, C.D., Simen, B.B., Hanczaruk, B., Nguyen, K.D., et al., 2009. Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing. Sci. Transl. Med. 1, 12ra23.

Calvo, S.E., Pagliarini, D.J., Mootha, V.K., 2009. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. U. S. A. 106, 7507-7512.

Campbell, P.J., Pleasance, E.D., Stephens, P.J., Dicks, E., Rance, R., Goodhead, I., Follows, G.A., Green, A.R., Futreal, P.A., Stratton, M.R., 2008. Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing. Proc. Natl. Acad. Sci. U. S. A. 105, 13081-13086.

Corcoran, M.M., Phad, G.E., Vazquez Bernat, N., Stahl-Hennig, C., Sumida, N., Persson, M.A., Martin, M., Karlsson Hedestam, G.B., 2016. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nat. Commun. 7, 13642.

Ebeling, M., Kung, E., See, A., Broger, C., Steiner, G., Berrera, M., Heckel, T., Iniguez, L., Albert, T., Schmucki, R., et al., 2011. Genome-based analysis of the nonhuman primate Macaca fascicularis as a model for drug safety assessment. Genome Res. 21, 1746-1756.

Elfakess, R., Dikstein, R., 2008. A translation initiation element specific to mRNAs with very short 50UTR that also regulates transcription. PLoS One 8, e3094.

Forthal, D.N., 2014. Functions of antibodies. Microbiol. Spectr. 2, 1-17.

Gadala-Maria, D., Yaari, G., Uduman, M., Kleinstein, S.H., 2015. Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles. Proc. Natl. Acad. Sci. U. S. A. 112, E862-E870.

Georgiou, G., Ippolito, G.C., Beausang, J., Busse, C.E., Wardemann, H., Quake, S.R., 2014. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat. Biotechnol. 32, 158-168.

Gibbs, R.A., Rogers, J., Katze, M.G., Bumgarner, R., Weinstock, G.M., Mardis, E.R., Remington, K.A., Strausberg, R.L., Venter, J.C., Wilson, R.K., et al., 2007. Evolutionary and biomedical insights from the rhesus macaque genome. Science 316, 222-234.

Gibbs, R.A., Weinstock, G.M., Metzker, M.L., Muzny, D.M., Sodergren, E.J., Scherer, S., Scott, G., Steffen, D., Worley, K.C., Burch, P.E., et al., 2004. Genome sequence of the brown Norway rat yields insights into mammalian evolution. Nature 428, 493-521.

Gibson, S.J., Bond, N.J., Milne, S., Lewis, A., Sheriff, A., Pettman, G., Pradhan, R., Higazi, D.R., Hatton, D., 2017. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains. Biotechnol. Bioeng. 114, 1970-1977.

Giudicelli, V., Chaume, D., Lefranc, M.P., 2005. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res. 33, D256-D261.

Glanville, J., Kuo, T.C., von Budingen, H.C., Guey, L., Berka, J., Sundar, P.D., Huerta, G., Mehta, G.R., Oksenberg, J.R., Hauser, S.L., et al., 2011. Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation. Proc. Natl. Acad. Sci. U. S. A. 108, 20066-20071.

Harbers, M., Kato, S., de Hoon, M., Hayashizaki, Y., Carninci, P., Plessy, C., 2013. Comparison of RNA- or LNA-hybrid oligonucleotides in templateswitching reactions for high-speed sequencing library preparation. BMC Genom. 14, 665.

Haryadi, R., Ho, S., Kok, Y.J., Pu, H.X., Zheng, L., Pereira, N.A., Li, B., Bi, X., Goh, L.T., Yang, Y., et al., 2015. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PLoS One 10, e0116878.

Hughes, M.J.G., Andrews, D.W., 1997. A single nucleotide is a sufficient 50 untranslated region for translation in an eukaryotic in vitro system. FEBS Lett. 414, 19-22.

Iacono, M., Mignone, F., Pesole, G., 2005. uAUG and uORFs in human and rodent 50untranslated mRNAs. Gene 349, 97-105.

Khan, T.A., Friedensohn, S., Gorter de Vries, A.R., Straszewski, J., Ruscheweyh, H.J., Reddy, S.T., 2016. Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting. Sci. Adv. 2, e1501371.

Klein, F., Gaebler, C., Mouquet, H., Sather, D.N., Lehmann, C., Scheid, J.F., Kraft, Z., Liu, Y., Pietzsch, J., Hurley, A., et al., 2012. Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein. J. Exp. Med. 209, 1469-1479.

Kreer, C., Doring, M., Lehnen, N., Ercanoglu, M.S., Gieselmann, L., Luca, D., Jain, K., Schommers, P., Pfeifer, N., Klein, F., 2020. Openprimer for multiplex amplification of highly diverse templates. J. Immunol. Methods 480, 112752.

Liao, B.Y., Zhang, J., 2008. Null mutations in human and mouse orthologs frequently result in different phenotypes. Proc. Natl. Acad. Sci. U. S. A. 105, 6987-6992.

Lingwood, D., McTamney, P.M., Yassine, H.M., Whittle, J.R., Guo, X., Boyington, J.C., Wei, C.J., Nabel, G.J., 2012. Structural and genetic basis for development of broadly neutralizing influenza antibodies. Nature 489, 566-570.

Lovett, P.S., Rogers, E.J., 1996. Ribosome regulation by the nascent peptide. Microbiol. Rev. 60, 366-385.

Matsui, M., Yachie, N., Okada, Y., Saito, R., Tomita, M., 2007. Bioinformatic analysis of post-transcriptional regulation by uORF in human and mouse. FEBS Lett. 581, 4184-4188.

Meng, W., Zhang, B., Schwartz, G.W., Rosenfeld, A.M., Ren, D., Thome, J.J.C., Carpenter, D.J., Matsuoka, N., Lerner, H., Friedman, A.L., et al., 2017. An atlas of B-cell clonal distribution in the human body. Nat. Biotechnol. 35, 879-884.

Mikocziova, I., Gidoni, M., Lindeman, I., Peres, A., Snir, O., Yaari, G., Sollid, L.M., 2020. Polymorphisms in human immunoglobulin heavy chain variable genes and their upstream regions. Nucleic Acids Res. 48, 5499-5510.

Neafsey, D.E., Galagan, J.E., 2007. Dual modes of natural selection on upstream open reading frames. Mol. Biol. Evol. 24, 1744-1751.

Parks, T., Mirabel, M.M., Kado, J., Auckland, K., Nowak, J., Rautanen, A., Mentzer, A.J., Marijon, E., Jouven, X., Perman, M.L., et al., 2017. Association between a common immunoglobulin heavy chain allele and rheumatic heart disease risk in Oceania. Nat. Commun. 8, 14946.

Ralph, D.K., Matsen, F.A. 4th, 2019. Per-sample immunoglobulin germline inference from B cell receptor deep sequencing data. PLoS Comput. Biol. 15, e1007133.

Scheid, J.F., Mouquet, H., Ueberheide, B., Diskin, R., Klein, F., Oliveira, T.Y., Pietzsch, J., Fenyo, D., Abadir, A., Velinzon, K., et al., 2011. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633-1637.

Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, M.C., Wardemann, H., 2008. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112-124.

Vergani, S., Korsunsky, I., Mazzarello, A.N., Ferrer, G., Chiorazzi, N., Bagnara, D., 2017. Novel method for high-throughput full-length IGHV-D-J sequencing of the immune repertoire from bulk B-cells with single-cell resolution. Front. Immunol. 8, 1157.

Vilela, C., McCarthy, J.E., 2003. Regulation of fungal gene expression via short open reading frames in the mRNA 50untranslated region. Mol. Microbiol. 49, 859-867.

Waterston, R.H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J.F., Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., An, P., et al., 2002. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520-562.

Watson, C.T., Glanville, J., Marasco, W.A., 2017. The individual and population genetics of antibody immunity. Trends Immunol. 38, 459-470.

Weinstein, J.A., Jiang, N., White 3rd, R.A., Fisher, D.S., Quake, S.R., 2009. Highthroughput sequencing of the zebrafish antibody repertoire. Science 324, 807-810.

Wellensiek, B.P., Larsen, A.C., Flores, J., Jacobs, B.L., Chaput, J.C., 2013. A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells. Protein Sci. 22, 1392-1398.

Yang, X., Wang, M., Shi, D., Zhang, Y., Zeng, H., Zhu, Y., Lan, C., Wu, J., Deng, Y., Guo, S., et al., 2021. Large-scale analysis of 2,152 dataset reveals key features of B cell biology and the antibody repertoire. Cell Rep. 35, 109110.

Zhang, W., Li, X., Wang, L., Deng, J., Lin, L., Tian, L., Wu, J., Tang, C., Yang, H., Wang, J., et al., 2019. Identification of variable and joining germline genes and alleles for rhesus macaque from B cell receptor repertoires. J. Immunol. 202, 1612-1622.

Zhang, Y., Xu, Q., Zeng, H., Wang, M., Zhang, Y., Lan, C., Yang, X., Zhu, Y., Chen, Y., Wang, Q., et al., 2020. SARS-CoV-2-, HIV-1-, Ebola-neutralizing and anti-PD1 clones are predisposed. bioRxiv. https://www.biorxiv.org/content/10.1101/2020.08.13.249086v2.

Zhou, Y., Liu, P., Gan, Y., Sandoval, W., Katakam, A.K., Reichelt, M., Rangell, L., Reilly, D., 2016. Enhancing full-length antibody production by signal peptide engineering. Microb. Cell Fact. 15, 47.

Relative Articles

Supplements(0)

Cited By

Proportional views