Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency

Abstract

Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.

Figure 1. Complementation analysis of Ade I by wt- and A291V-hADSL plasmids

Ade I cells were transfected with wt (hADSL-pCR3.1) or A291V (pLKV1) hADSL plasmids. After 24–48 hr, cells were split into fresh media containing Geneticin. Cells were split every 48–72 hr into media containing Geneticin for several days. Geneticin resistant clones were isolated and grown in the presence (+) or absence (−) of adenine. Cells were fixed and stained in 1% crystal violet (w/v) in 50:50 (v/v) dH₂O: methanol.

Figure 2. Enzymatic Assay of CHO Cell Lysates for ADSL Activity

CHO cell lysates from untransfected K1 and Ade I (open and closed diamonds, respectively) and Ade I cells transfected with wild-type hADSL (hADSL-pCR3.1 clones 1.1a and 2.1c, open and closed triangles, respectively) or A291V hADSL (pLKV1 clones 1.1b and 2.1d, open and closed squares, respectively) were assayed for ADSL activity. Data represent one of three independent assays.

Figure 3. qPCR analysis of ADSL mRNA levels in CHO-K1 and AdeI cells

Total RNA was isolated and cDNA prepared from CHO cells (K1 and AdeI). Normalized fold expression levels of ADSL transcripts in K1 and AdeI was determined by qPCR, in triplicate, using ADSL gene specific primers and 18s ribosomal RNA reference gene primers. Relative expression levels were determined using the ΔΔCt method.

Figure 4. Expression of hADSL mRNA in transfected Ade I cells

(Upper panel) Total RNA was isolated and cDNA prepared from Ade I transfected with either wt (hADSL-pCR3.1) or A291V (pLKV1) hADSL plasmids. PCR was performed on cDNA from two independently-isolated clones for wt (1.1a and 2.1c) and A291V (1.1b and2.1d). Untransfected Ade I and CHO cDNA were used as negative controls. A no template control (NT) was also subjected to PCR. cDNA prepared from HeLa total RNA was used as a positive control. (Lower panel) The CHO β-actin gene was amplified by PCR to confirm RNA integrity.

Figure 5. Western blot of AdeI transfectants

Fifteen µg of protein from CHO cell lines was analyzed by western blotting using anti-ADSL and anti-B-actin polyclonal antibodies (see Materials and Methods). Each sample lane was loaded with with 15 ug protein. The lanes are: 1) MW (MagicMark XP, Invitrogen), 2) CHO-K1, 3) AdeI, 4) PCR 3.1 2.1c, 5) pLKV1 1.1b, and 6) Recombinant ADSL (Kmoch Lab).

Figure 6. Structural model of human ADSL

(A) Four ADSL subunits forming native homotetramer are shown in different colors. All four of the enzyme active sites are highlighted by presence of one of the ADSL substrates, S-AMP (green spheres). A291V residues and adjacent flexible loops lining the substrate channels of the enzyme are present between Phe282 and Pro293 residues shown in yellow. (B) Detail of one of the active sites.