Advertisement

Human Genetics

, Volume 88, Issue 3, pp 313–319 | Cite as

Cloning of the human α2-macroglobulin gene and detection of mutations in two functional domains: the bait region and the thiolester site

  • W. Poller
  • J.-P. Faber
  • G. Klobeck
  • K. Olek
Original Investigations

Summary

Overlapping genomic clones of the human α2-macroglobulin (α2M) gene were isolated from a cosmid library and were used to map 80 kb of the chromosomal region of this gene. Fragments carrying the two exons encoding the bait region and the exon encoding the thiolester site were partially sequenced and PCR primers were designed for the amplification of both functional domains. By direct genomic sequencing of these domains in 30 healthy individuals and in 30 patients with chronic lung disease three mutations were detected. The first was a sequence polymorphism occurring near the thiolester site of the gene, changing Val1000(GTC) to Ile1000(ATC), with allele frequencies of 0.30 (GTC) and 0.70 (ATC), respectively. No difference of α2M serum levels was observed for these two alleles. The second mutation occured within the thiolester site of one patient, changing Cys972(TGT) to Tyr972(TAT). Since activation of the internal thiolester formed between Cys972 and Gln975 in each of the subunits of the tetrameric α2M is involved in the covalent cross-linking of the activating proteinase, this mutation is predicted to interfere with α2M function. The α2M serum level was within the normal range in this patient. In one healthy individual we detected an alteration of the bait region sequence, which is usually encoded by two different exons separated by an intron of size 1.6kb. In this individual, PCR amplification of genomic DNA using the bait region primers produced the common fragment of size 1.8 kb and an additional variant fragment of size 0.23kb. This finding, and the genomic sequencing data of this individual, indicate that he carries two different alleles of the α2M gene: one with the regular structure (bait exon I-intron-bait exon II), the other with the two bait exons fused into one. Direct genomic sequencing of the two α2M functional domains is a useful tool for the detection of the genetic, and possibly the functional, heterogeneity of α2M. This, in turn, may provide some insight into the hitherto unknown physiological role(s) of α2M, by studying in vivo effects of naturally ocurring mutations of the gene.

Keywords

Healthy Individual Lung Disease Chromosomal Region Region Sequence Functional Domain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bretaudiere J-P, Tapon-Bretaudiere J, Stoops JK (1988) Structure of native α2-macroglobulin and its transformation to the protease bound form. Proc Natl Acad Sci USA 85:1437–1441Google Scholar
  2. Brissenden JE, Cox DW (1982) α2-macroglobulin production by cultured human fibroblasts. Somat Cell Genet 8:289–305Google Scholar
  3. Crystal RG (1990) α1-antitrypsin deficiency, emphysema, and liver disease. J Clin Invest 85:1343–1352Google Scholar
  4. Delacroix DL, Marchandise FX, Francis C, Sibille Y (1985) α2-macroglobulin, monomeric and polymeric immunoglobulin A, and immunoglobulin M in bronchoalveolar lavage. Am Rev Respir Dis 132:829–835Google Scholar
  5. Devriendt K, Zhang J, Leuven F van, Berghe H van den, Cassiman J-J, Marynen P (1989) A cluster of α2-macroglobulin-related genes (α2M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and an α2-macroglobulin pseudogene. Gene 81:325–334Google Scholar
  6. Feldman SR, Gonias SL, Pizzo SV (1985) Model of α2-macroglobulin structure and function. Proc Natl Acad Sci USA 82:5700–5704Google Scholar
  7. Gibson TJ, Coulson AR, Sulston JE, Little P (1987) Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promotors within the insert: effect on DNA yield and cloned insert frequency. Gene 53:275–281Google Scholar
  8. Hattori M, Kusukabe S-I, Ohgusu H, Tsuchiya Y, Ito Y, Sakaki Y (1989) Structure of the rat α2-macroglobulin-coding gene. Gene 77:333–340Google Scholar
  9. Ish-Horowitz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998Google Scholar
  10. Kan C-C, Solomon E, Belt KT, Chain AC, Hiorns LR, Fey G (1985) Nucleotide sequence of cDNA encoding human α2-macroglobulin and assignment of the chromosomal locus. Proc Natl Acad Sci USA 82:2282–2286Google Scholar
  11. Marynen P, Devriendt K, Van Den Berghe H, Cassiman JJ (1990) A genetic polymorphism in a functional domain of human pregnancy-zone protein: the bait region. FEBS Lett 262:349–352Google Scholar
  12. Mosher DF, Wing WA (1976) Synthesis and secretion of α2-macroglobulin by cultured human fibroblasts. J Exp Med 143:462–467Google Scholar
  13. Munck Petersen C, Christiansen BS, Heikendorff L, Ingerslev J (1988a) Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture. Eur J Clin Invest 18:543–548Google Scholar
  14. Munck Petersen C, Christiansen B, Jensen PH, Moestrup SK, Gliemann J, Sottrup-Jensen L, Ingerslev J (1988b) Human hepatocytes exhibit receptors for α2-macroglobulin and pregnancy-zone protein — proteinase complexes. Eur J Clin Invest 18:184–190Google Scholar
  15. Petersen CM, Ejlersen E, Hansen PW, Gliemann J (1987) Binding of α2-macroglobulin-trypsin complex to human monocytes in culture. Scand J Clin Lab Invest 47:55–61Google Scholar
  16. Poller W, Barth J, Voss B (1989) Detection of an alteration of the α2-macroglobulin gene in a patient with chronic lung disease and serum α2-macroglobulin deficiency. Hum Genet 83:93–96Google Scholar
  17. Sottrup-Jensen L, Stepanik TM, Kristensen T, Wierzbicki DM, Jones CM, Lonblad PM, Magnusson S, Petersen TE (1984) Primary structure of human α2-macroglobulin. V. The complete structure. J Biol Chem 259:8318–8327Google Scholar
  18. Sottrup-Jensen L, Sand O, Kristensen L, Fey GH (1989) The α2-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian α2-macroglobulins. J Biol Chem 264:15781–15789Google Scholar
  19. Starkey PM, Barrett AJ (1977) α2-macroglobulin, a physiological regulator of proteinase activity. In: Barrett AJ (ed) Proteinases in mammalian cells and tissues. North-Holland, New York, pp 663–696Google Scholar
  20. Sykes BC (1983) DNA in heritable disease. Lancet II:787–788Google Scholar
  21. Van Leuven F, Marynen P, Sottrup-Jensen L, Cassiman J-J, Van Den Berghe H (1986a) The receptor binding domain of human α2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. J Biol Chem 261:11369–11373Google Scholar
  22. Van Leuven F, Cassiman J-J, Van Berghe H (1986b) Human pregnancy-zone protein and α2-macroglobulin: high affinity binding to the same receptor on fibroblasts and characterization by monoclonal antibodies. J Biol Chem 251:16622–16625Google Scholar
  23. Wewers MD, Casolaro MA, Sellers S, Swayze SC, McPhaul KM, Crystal RG (1987) Replacement therapy for alpha1-antitrypsin deficiency associated with emphysema. N Engl J Med 316:1055–1062Google Scholar
  24. White R, Janoff A, Godfrey HP (1980) Secretion of α2-macroglobulin by human alveolar macrophages. Lung 158:9–14Google Scholar
  25. Wong C, Dowling CE, Saiki RK, Higuchi RG, Erlich HA, Kazazian HH (1987) Characterization of β-thalassemia mutations using direct genomic sequencing of amplified single-copy DNA. Nature 330:384Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • W. Poller
    • 1
  • J.-P. Faber
    • 2
  • G. Klobeck
    • 3
  • K. Olek
    • 2
  1. 1.Medizinische Universitätsklinik und Poliklinik, Klinikum BergmannsheilRuhr-UniversitätBochumGermany
  2. 2.Institut für Klinische Biochemie der UniversitätBonnGermany
  3. 3.Institut für Physiologische ChemieLudwig-Maximilians-UniversitätMünchenGermany

Personalised recommendations