Erythroid pyrimidine 5′-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia

Abstract

A characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. The p5n-I expression is induced (1) IntroductionDuring vertebrate embryonic/early fetal development, the major portion of circulating red blood cells (RBCs) is not fully mature. Therefore, processes such as organelle breakdown and nuclear shutdown must be completed while the RBCs already carry out their gas transport function. To study the coupling of final RBC maturation and function, the chicken embryo is an excellent model (for review see Dragon and Baumann 1 ); all important respiratory parameters that govern gas transport (pH, Pco 2 , Po 2 ) 2,3 have been established throughout development, and the embryonic/fetal stages are easily accessible. It has long been known that in the late chicken embryo, the circulating RBCs adjust their hemoglobin-oxygen affinity to the continuously falling Po 2 by changing their organic phosphate pattern; while in the first two thirds of development, adenosine triphosphate (ATP), uridine triphosphate (UTP), and cytidine triphosphate (CTP) determine the hemoglobin-oxygen affinity, 2,3-bisphosphoglycerate (2,3-BPG) becomes the major organic phosphate of the RBCs in the last week of incubation. [4][5][6][7] The exchange of nucleotides by 2,3-BPG is promoted by the embryonic hormones norepinephrine (NE) and adenosine [8][9][10] and is efficiently blocked by transcriptional inhibition. 11 The 2 hormones that are released by the increasing hypoxia during embryonic development 9,10 also control other processes of late erythroid maturation; they induce regulatory genes (tob, ífr1, fos, hsp70) 12,13 as well as carbonic anhydrase (CAII) and pyrimidine 5Ј-nucleotidase (P5N-I)-2 important erythroid enzymes. 8,9,12,[14][15][16] In humans, the P5N-I enzyme is critically involved in the degradation of pyrimidine nucleotides and ribosomal RNA during final erythroid maturation because an enzyme deficiency causes the accumulation of ribosomal structures and pyrimidine trinucleotides (UTP, CTP). 17,18 Likewise, the chicken embryonic RBCs liberate cellpermeable pyrimidine nucleosides upon hormonal induction of the enzyme, and P5N-I enzyme activity...

Functional analysis of pyrimidine 5′-nucleotidase mutants causing nonspherocytic hemolytic anemia

“…11 The mutation targets an amino acid residue that falls in a region highly conserved among species. 5,20 Unlike the other 3 mutants here considered, N179S enzyme turned out to display a stability comparable to the wild-type enzyme. Conversely, it exhibited a greatly impaired kinetic behavior.…”

“…5 The reduced heat stability (t 1/2 at 46°C, 3.5 minutes; T 50 , 10°C lower than the wild type) together with the decreased catalytic efficiency, especially toward UMP (about 25 times), is likely to be the reason for the low activity found in patients. Moreover, with Asp87 being a well-conserved amino acid, 5,20 we suggest that this residue might be part of the active site.…”

In embryonic chicken erythrocytes actively transcribed alpha globin genes are not associated with the nuclear matrix

“…To induce erythroid differentiation, HD3 cells at a density of 8 Â 10 5 cells/ml were incubated in the above medium additionally containing 10 mM HEPES (pH 8.0) and 20 mM iso-H-7 (1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride, Fluka) at 428C in 100% air atmosphere [Nicolas et al, 1991]. Ten-day chicken embryonic red blood cells (RBC) were prepared as described previously [Mass et al, 2003].…”

Hereditary pyrimidine 5′‐nucleotidase deficiency: from genetics to clinical manifestations

“…The major role of P5′N‐1 is found in the catabolism of the pyrimidine nucleotides, mainly resulting from ribosomal RNA degradation during final erythroid maturation (Paglia & Valentine, 1975; Mass et al , 2003; Rees et al , 2003). The pyrimidine nucleotides are not used by mature erythrocytes and need to be transported out of the cells to prevent their accumulation.…”

“…Asn179Ser enzyme displays an extreme reduction of the catalytic efficiency (more than two to three orders of magnitude), but no alteration in thermostability. As Asn belongs to a region highly conserved among species (Marinaki et al , 2001; Mass et al , 2003), it is likely that P5′N‐1 deficiency is related to an inactive enzyme in which a functionally crucial residue, directly involved in catalysis and substrate binding, has been replaced.…”