Elsevier

Gene

Volume 308, 10 April 2003, Pages 95-101
Gene

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

https://doi.org/10.1016/S0378-1119(03)00470-0Get rights and content

Abstract

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc langurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions.

Introduction

Angiogenin (ANG) was originally isolated from human tumor cell-conditioned medium based on its ability to stimulate formation of new blood vessels (angiogenesis) (Fett et al., 1985). It was subsequently found to belong to the RNase A superfamily by sequence homology (Kurachi et al., 1985, Strydom et al., 1985) and was named RNase 5 (reviewed in Strydom, 1998). RNase A superfamily has 8 members in humans (Zhang et al., 2002a) and they exhibit drastically different physiological functions, albeit with the common ribonuclease activity (D'Alession and Riordan, 1996, Beintema and Kleineidam, 1998). ANG is the only member of the superfamily known to be angiogenic. Although the ribonuclease activity of ANG is about a million times lower than that of pancreatic RNase, the prototype of the superfamily, the ribonuclease activity is required for the angiogenic activity (Shapiro et al., 1986). The molecular mechanism of the angiogenic activity has not been fully elucidated, though a working model has been proposed (Vallee and Riordan, 1997). ANG has elevated expression in various tumors, including breast, pancreatic, gastric, colorectal, and urothelial cancers, and the angiogenic activity is related to cancer progression (Shimoyama et al., 1996, Shimoyama et al., 1999, Montero et al., 1998, Miyake et al., 1999, Shimoyama and Kaminishi, 2000). Not surprisingly, ANG antagonists have the ability to inhibit cancer growth in vivo (Olson et al., 1994, Olson et al., 1995, Piccoli et al., 1998, Kao et al., 2002). In addition, several authors suggested the role for ANG during pregnancy in tissue vascularization of the developing embryo (Hayashi et al., 2000, Koga et al., 2000, Malamitsi-Puchner et al., 2000). Given these properties, it becomes increasingly important to understand the physiological function of angiogenin. In principle, the function of ANG can be studied in model organisms such as the mouse. The phenotype of the mouse with the ANG gene deleted can reveal the physiological roles of ANG. The situation, however, is complicated by the fact that there is only one ANG gene in the human, but three in the mouse (Brown et al., 1995). Phylogenetic analyses suggested that the three ANG genes of the mouse were generated from two rodent-specific gene duplications (Strydom, 1998, Zhang and Rosenberg, 2002). Mouse ANG1 and ANG3 are angiogenic whereas ANG2 is not (Nobile et al., 1996, Fu et al., 1999). Because it is unknown whether the physiological function of ANG is related to its angiogenic activity, it seems necessary to knockout all three ANG genes of the mouse to study the normal function of ANGs. Furthermore, due to the rodent-specific gene duplications and possible functional changes after the duplications, it is unclear whether the function of human ANG can be adequately inferred from the knockout mice. After all, it is the functional information of human ANG that is needed for understanding its role in tumor-growth promotion. Given these facts, it would be ideal to use a primate model rather than a rodent model to study ANG, as primates are not known to have more than one ANG gene (Zhang and Rosenberg, 2002). In an evolutionary survey, we inadvertently discovered that the ANG gene of a leaf-eating monkey is naturally inactivated. Here we report this finding as well as an evolutionary analysis of the pseudogenization event. We propose that a study of this monkey species may reveal useful information on the physiological function of human ANG.

Section snippets

DNA sequencing

The ANG gene has only one coding exon, which was amplified from genomic DNAs of six species belonging to the subfamily Colobinae of Old World monkeys by PCR with primers ANG-5 (5′-GTGTTGGAAGAGATGGTGATGGGC-3′) and ANG-3 (5′-AGCACTTGACCAGGGGCCCGCTGGTTA-3′). The first four codons of the ANG gene were encoded in the ANG-5 primer. The six species are the douc langur (Pygathrix nemaeus), Tonkin snub-nosed monkey (Rhinopithecus avunculus), Biet's snub-nosed monkey (Rhinopithecus bieti), golden

ANG pseudogene of the douc langur

We previously demonstrated that the ANG gene evolves rapidly under diversifying positive selection from a study of 11 noncolobine primates (Zhang and Rosenberg, 2002). In an expanded evolutionary survey of the gene among different primates, we inadvertently found in the douc langur (P. nemaeus) a one-nucleotide deletion in the ANG coding region. The deletion occurs at the second position of the sixth codon of the mature peptide, and shifts the open reading frame (Fig. 1). This results in the

Discussion

In this study, we showed that the ANG gene of the douc langur P. nemaeus was inactivated by a one-nucleotide deletion in its coding region. We believe that this pseudogene is the sole ANG gene in the douc langur, because our extensive PCR-cloning-sequencing experiments resulted in only one gene. Furthermore, a search in the human genome sequence identified only one ANG gene (Zhang and Rosenberg, 2002, Zhang et al., 2002a) and previous phylogenetic analyses of the ANG sequences from 11

Acknowledgements

We thank Jaap Beintema, David Webb, and two anonymous reviewers for their comments on an early version of the manuscript. This work was supported by a startup fund from the University of Michigan to J.Z. and research grants from the National Institutes of Health (R01-GM67030) to J.Z., Natural Science Foundation of China to Y.P.Z., and Chinese Academy of Sciences (KSCX2-1-05) to Y.P.Z.

References (41)

  • W.E Brown et al.

    The mouse angiogenin gene family: structures of an angiogenin-related protein gene and two pseudogenes

    Genomics

    (1995)
  • J.J Beintema et al.

    The ribonuclease A superfamily: general discussion

    Cell. Mol. Life Sci.

    (1998)
  • G D'Alession et al.

    RNases, Structures and Functions

    (1996)
  • E Delson

    Evolutionary history of the colobine monkeys in paleoenvironmental perspectives

  • J Felsenstein

    Confidence limits on phylogenies: An approach using the bootstrap

    Evolution

    (1985)
  • J.W Fett et al.

    Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells

    Biochemistry

    (1985)
  • X Fu et al.

    mAngiogenin-3, a target gene of oncoprotein E2a-Pbx1, encodes a new angiogenic member of the angiogenin family

    Growth Factors

    (1999)
  • K Hayashi et al.

    Serum angiogenin levels during menstrual cycle and pregnancy

    Gynecol. Obstet. Invest.

    (2000)
  • R.Y Kao et al.

    A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity

    Proc. Natl. Acad. Sci. USA

    (2002)
  • M Kimura

    A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences

    J. Mol. Evol.

    (1980)
  • K Koga et al.

    Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia

    J. Clin. Endocrinol. Metab.

    (2000)
  • S Kumar et al.

    A molecular timescale for vertebrate evolution

    Nature

    (1998)
  • S Kumar et al.

    MEGA2, Molecular Evolutionary Genetic Analysis

    (2000)
  • K Kurachi et al.

    Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

    Biochemistry

    (1985)
  • A Malamitsi-Puchner et al.

    Angiogenic factors in the perinatal period: diversity in biological functions reflected in their serum concentrations soon after birth

    Ann. N. Y. Acad. Sci.

    (2000)
  • H Miyake et al.

    Increased angiogenin expression in the tumor tissue and serum of urothelial carcinoma patients is related to disease progression and recurrence

    Cancer

    (1999)
  • S Montero et al.

    Angiogenin expression and prognosis in primary breast carcinoma

    Clin. Cancer Res.

    (1998)
  • M Nei et al.

    Evolution by the birth-and-death process in multigene families of the vertebrate immune system

    Proc. Natl. Acad. Sci. USA

    (1997)
  • V Nobile et al.

    Characterization of mouse angiogenin-related protein: implications for functional studies on angiogenin

    Proc. Natl. Acad. Sci. USA

    (1996)
  • R.M Nowak

    Walker's Mammals of the World

    (1999)
  • Cited by (12)

    • The genomic context of retrocopies increases their chance of functional relevancy in mammals

      2020, Genomics
      Citation Excerpt :

      Contrary to the theoretical expectation that any gene can form a pseudogene, differences have been reported in the relative proportion, from gene to gene [18]. Housekeeping genes, highly expressed genes in germ-line cells and those participating in basic metabolic regulations show multiple corresponding pseudogenes [16,19–21]. This may be due to high expression levels of these genes, thus increasing their likelihood to accumulate mutations [22] or to be retro-transposed into the genome [23].

    • Angiogenesis

      2008, Microcirculation
    • Positive selection in the evolution of cancer

      2006, Biological Reviews of the Cambridge Philosophical Society
    • The Ribonuclease A Superfamily: Not Only a Class of Enzymes for RNA Degradation

      2023, Chinese Journal of Biochemistry and Molecular Biology
    View all citing articles on Scopus

    Sequence data from this article have been deposited with GenBank under accession numbers AY221128–221134.

    View full text