Elsevier

Gene

Volume 197, Issues 1–2, 15 September 1997, Pages 389-398
Gene

Isolation and nucleotide sequence of human liver glycogen debranching enzyme mRNA: identification of multiple tissue-specific isoforms

https://doi.org/10.1016/S0378-1119(97)00291-6Get rights and content

Abstract

Glycogen storage disease type III (GSD-III) is caused by a deficiency of glycogen debranching enzyme (AGL) activity. Patients are found to have deficient AGL activity in both muscle and liver, and also enzyme deficiency in the liver, but not in muscle. To determine the molecular basis of enzymatic variability in GSD-III and to elucidate the mechanism for control of tissue-specific expression of AGL, we previously cloned and sequenced the human muscle AGL cDNA. Here we report the isolation and nucleotide sequence of liver AGL cDNA and the tissue distribution of the isoform mRNAs. The predominant form of human liver AGL cDNA (isoform 1) contained 400 bp of 5′ untranslated region, 4596 bp of coding region, and 2371 bp of 3′ untranslated region. The liver AGL mRNA sequence was identical to the previously published muscle sequence (isoform 5) for most of the length, except for the 5′ end, in which the liver sequence diverged completely from the muscle sequence. The divergence began with the transcription start point and extended 82 nucleotides downstream from the translation initiation codon. Six isoforms of AGL mRNA were identified and sequenced from liver and muscle. These isoforms differed only at the 5′ end. Tissue distribution studies showed that liver, kidney and lymphoblastoid cells expressed predominantly isoform 1; whereas muscle and heart expressed not only isoform 1, but also muscle-specific isoform mRNAs (isoforms 2, 3 and 4). Defining tissue-specific AGL isoform mRNAs is an important step toward understanding the molecular basis of enzymatic variability in GSD-III.

Introduction

Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting as 1,4-α-d-glucan:1,4-α-d-glucan 4-α-d-glycosyltransferase (EC 2.4.1.25) and amylo-1,6-glucosidase (EC 3.2.1.33) in glycogen degradation (Gordon et al., 1972; White and Nelson, 1974; Taylor et al., 1975). The two activities are believed to reside on a single polypeptide chain with a molecular mass of about 165 kDa. The debranching enzyme, together with phosphorylase are responsible for complete degradation of glycogen. Liver and muscle are the two major organs most active in glycogen metabolism. The primary function of glycogen in these organs is different; in muscle, glycogen provides a local fuel store for short-term energy consumption; in liver, glycogen maintains glucose homeostasis. Regulation of glycogen metabolism is conferred, at least in part, by tissue-specific isoenzymes encoded by different genes. For example, liver, muscle and brain phosphorylases are products of three distinct genes, each under separate genetic control (for review see Newgard et al., 1989). The structure and function of all the phosphorylase isoenzymes have been extensively characterized. The AGL, however, has only been studied in detail in muscle, and little is known of the AGL in liver. At the protein level, it appears that there are no tissue-specific AGL isoenzymes present in different tissues (Gordon et al., 1972; Chen et al., 1987; Ding et al., 1990).

Genetic deficiency of AGL activity, known as glycogen storage disease type III (GSD-III), causes an incomplete glycogenolysis which results in accumulation of glycogen with abnormally short outer branches in various organs, mostly in liver and skeletal muscle. Clinically, patients with GSD-III manifest with hepatomegaly, hypoglycemia, short stature, and variable myopathy (Chen and Burchell, 1995). The variable phenotype is explained by differences in tissue-specific expression of the defective enzyme. Most commonly the enzyme is deficient in both liver and muscle (GSD-IIIa). However, sometimes AGL is deficient only in liver and activity in muscle is normal (GSD-IIIb). In rare cases, selective loss of only one of the two AGL activities (glucosidase (GSD-IIIc), or transferase (GSD-IIId)) has been demonstrated (Van Hoof and Hers, 1967; Brown and Brown, 1968)).

We have previously cloned and sequenced a human muscle debranching enzyme cDNA (Yang et al., 1992) which has a 4545 bp coding region and a 2371 bp 3′-untranslated region. A rabbit muscle AGL cDNA was subsequently cloned (Liu et al., 1993). The sequence homology between human and rabbit is high throughout the majority of the gene, except for sequence diversity in the extreme 5′-region. The human gene is localized to chromosome 1p21 (Yang-Feng et al., 1992). To further investigate whether the liver AGL mRNA is identical to or is a gene product distinct from the muscle mRNA, and to elucidate mechanistically how AGL, a monomeric protein normally expressed in all tissues, can change expression in different tissues, we cloned and sequenced the human liver AGL mRNA. Multiple tissue-specific isoforms of AGL mRNAs were found, differing only in the 5′-end.

Section snippets

Total cellular RNA and poly(A)+ RNA extraction

Total cellular RNAs were extracted from normal human liver, skeletal muscle, heart, kidney and lymphoblastoid cells using the acid guanidinium–phenol–chloroform method (Chomczynski and Sacchi, 1987) or with Trizol® (Gibco-BRL, Grand Island, NY, USA) following manufacturer's instructions. Poly(A)+ RNA was purified twice by oligo(dT)-cellulose spin columns (Pharmacia, Piscataway, NJ, USA) following the manufacturer's instructions.

Northern blot analysis

A nylon membrane blotted with 2 μg of poly(A)+ RNAs from normal

Tissue distribution of AGL mRNA

Northern blot analysis was performed to investigate whether muscle AGL cDNA cross-hybridized to mRNA molecules from other tissues, and to determine the size and relative abundance of AGL mRNA in various tissues (Fig. 1). RNA molecules of the same apparent size as those in skeletal muscle were detected in heart, brain, placenta, liver and pancreas. The signal intensity was strikingly higher in skeletal muscle and heart than in other tissues. The Northern blot was probed to assess the quality and

Discussion

We have isolated and sequenced the human glycogen debranching enzyme (AGL) mRNA from liver using AGL cDNA isolated from muscle as a probe. The liver cDNA was identical to the previously published muscle sequence, except for the 5′-untranslated region and the first 82 nucleotides downstream from the putative translation initiation codon in exon 3. Several facts from the studies of enzyme activity, size, and antigenicity of AGL protein indicate that the AGL protein of liver is very similar, if

Acknowledgements

This work was supported by grants from National Institute of Health DK39078 (to Y.T.C.) and M01-RR30 (National Center for Research Sources, General Clinical Research Centers Program), and Muscular Dystrophy Association.

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Present address: Institute of Metabolic Disease, Baylor University Medical Center, Dallas, TX, USA.

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