Human adenosine deaminase. cDNA and complete primary amino acid sequence.

P E Daddona,D S Shewach,W N Kelley,P Argos,A F Markham,S H Orkin

doi:10.1016/s0021-9258(20)71325-6

Abstract

A previously cloned partial adenosine deaminase cDNA insert (0.8 kilobase) was used to clone additional nucleotide sequences from human HPB ALL cDNA libraries. cDNA encompassing the entire coding, and 3'-untranslated regions as well as nearly all of the 5'-untranslated region was obtained. The complete amino acid sequence of the enzyme deduced from the cDNA sequence and protein sequencing consists of 362 amino acids, excluding the initiator Met, and accounts for Mr = 40,638. Secondary structure predictions assign adenosine deaminase to the alpha/beta class of proteins. Northern blot analysis with a cDNA probe showed adenosine deaminase mRNA to be present in normal to above normal amounts in B-lymphoblasts derived from adenosine deaminase-deficient patients with severe combined immunodeficiency disease. Knowledge of the cDNA and primary amino acid sequence of adenosine deaminase will be pivotal in further defining the genetic abnormality and its functional consequences in adenosine deaminase expression defects.

Highlights

From the $Departmentsof Internal Medicine and Biological Chemistry, University of MichiganMedical Center, Ann Arbor, Michigan 48109, the Q Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,11 Imperial
The complete aminodeaminase purified from humanerythrocytes is a soluble acid sequence of the enzyme deduced from the cDNA monomeric protein with an estimated M, = 38,000 [11, 12]
Apartial genetic deficiency of adenosine cDNA and primary amino acid sequence of adenosine deaminase is associated with undetectable enzyme activity in deaminase will be pivotal in further defining the ge- senescenterythrocytes and with partial activity (1-20%of netic abnormality and its functional consequences in noimal) in peripheral lymphocytes; these subjects are immuadenosine deaminaseexpression defects

Summary

Human Adenosine Deaminase cDNA AND COMPLETE PRIMARY AMINO ACID SEQUENCE*

18).Recent preliminary studiessuggest that anabove normal level of adenosine deaminase mRNA is present in a B-lymphoblast cell line derived from a severely and from a partially adenosine deaminase-deficient subject In both subjects, the mutation appears to be associated with an unstable adenosine deaminase protein [19, 20]. We have previously reported the isolation of a partial human adenosine deaminase cDNA gene sequence (0.8-kilobase insert) from a T-cell library using amino acid sequence data to construct a mixed oligonucleotide probe [21]. RNA Isolation and Quantitation-Total RNA was isolated from cultured human B-lymphoblast cell lines derived from normal individuals (GM 333 and GM 558), from patients with adenosine deaminase deficiency and severe combined immunodeficiencydisease The two N- andthe two C-terminal values were respectively determined from the lines calculated for points 1 to 5 and 358 to 362

RESULTS AND DISCUSSION

Adenosine Deaminase Sequence

TABLEI Amino acid composition of human adenosine deaminase

Amino acid region t