Grycobiotogy vol. 2 no. 4 pp. 383-393, 1992

Cloning and expression of the murine gene and chromosomal location of the human gene encoding Af-acetylglucosaminyltransferase I

Ravindra Kumar1*, Jing Yang1*, Roger L.Eddy2, Mary G.Byers2, Thomas B.Shows2 and Pamela Stanley1-3 'Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461 and 2Depanment of Human Genetics, Roswell Park Cancer Institute, 666 Elm Sreet, Buffalo, NY 14263, USA 'To whom correspondence should be addressed

Results Sequence of a mouse cDNA encoding GlcNAc-TI activity

Key words: bacterial expression/chromosomal mapping/cloned glycosyltransferase/mouse gene expression Introduction The glycosyltransferase UDP-A'-acetylglucosamine: a-3-Dmannoside |3-l,2-A'-acetylglucosaminyltransferase I, termed N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), is a medial Golgi enzyme that initiates complex and hybrid These authors contributed equally to the cloning and expression studies. c, Oxford University Press

Four cDNAs that conferred GlcNAc-TI activity upon transfection into Lecl CHO cells were previously isolated from an F9 teratocarcinoma cell cDNA library following screening with a unique probe for the human GlcNAc-TT gene (Kumar et al, 1990). The sequence of a clone with the largest insert size is presented in Figure 1. The total of 2694 nucleotides included an open reading frame (ORF) from nucleotides 364 to 1707. The translation start site is predicted to occur at die ATG at nucleotides 364-366 which was the only one of the four ATGs in the 5' sequence to encode a continuous amino acid sequence of significant length. The context of this ATG conforms to the 383

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A mouse cDNA clone previously isolated from an F9 teratocarcinoma cell library and shown to confer N-acetylglucosaminyltransferase I (GlcNAc-TI) activity on Lecl Chinese hamster ovary (CHO) cell transfectants [Kumar, R., Yang,J., Larsen,R.D. and Stanley,P. (1990) Proc. Natl. Acad Sci. USA, 87, 9948-9952] has been sequenced. The nucleotide and deduced amino acid sequences are highly homologous to previously described human and rabbit GlcNAc-TI cDNAs. A 1250 bp portion of the mouse cDNA encoding all but the first 34 amino acids of the deduced protein sequence was inducibly expressed in Escherichia coli and gave rise to a prominent fusion protein of mol. wt ~ 45 kDa whose presence correlated with high levels of GlcNAcTI activity in cell lysates. Probes generated from the cDNA were used to show that the GlcNAc-TT gene is present in a single copy in mammals and that a homologous gene was not detectable (under low-stringency hybridization conditions) in DNA from yeast, sea urchin, Drosophila or Chaenorhabditis elegans. Genomic DNA clones that hybridized to probes generated from the GlcNAc-TI cDNA were isolated from a mouse liver library. Restriction analyses, Southern hybridization and DNA sequence analyses of subcloned genomic DNA fragments and a polymerase chain reaction (PCR) product provided evidence that the coding and 3' untranslated regions of the cDNA reside in a single exon. However, the mouse GlcNAc-TI gene (Mgat-1) includes at least one additional exon 5' of the coding region. Southern analyses of DNA from mouse-human somatic cell hybrids and in situ hybridization were used to locate the human GlcNAc-TI gene (MGAT-1) between positions q31.2 and q31.3 on chromosome 5, a region of chromosome 5 that is syntenic with a region of mouse chromosome 11. Northern analyses of adult mouse tissues revealed two GlcNAc-TI gene transcripts that are differentially expressed in different tissues.

N-linked carbohydrate synthesis (Kornfeld and Kornfeld, 1985). It is, therefore, a key enzyme in the regulation of expression of complex carbohydrates in mammalian cells. In its absence, the biosynthesis of complex carbohydrates stops at the intermediate Man5GlcNAc2Asn which becomes the structure present at all N-linked glycosylation sites that would normally carry complex or hybrid moieties. Chinese hamster ovary (CHO) mutant cell lines that lack GlcNAc-TI activity (Gottlieb et al., 1975; Stanley et al., 1975) are perfectly viable and grow with a normal doubling time in suspension as well as in monolayer culture (Stanley, 1989). Thus, it is clear that complex N-linked carbohydrates are not essential for the survival or proliferation of mammalian cells in culture. However, it is expected that these classes of N-linked carbohydrates, which are known to vary specifically in structure during development and differentiation (Muramatsu, 1988), will be found to be important to the viability or development of a mammalian organism. To test this hypothesis, it will be necessary to alter GlcNAc-TI expression in an experimental animal model such as the mouse. In previous studies, this laboratory used an expression cloning strategy to isolate a unique fragment of the human GlcNAcTI gene for use as a probe to obtain human and mouse cDNAs that conferred GlcNAc-TI activity on transfected Lecl CHO cells (Kumar and Stanley, 1989; Kumar et al, 1990). A rabbit GlcNAc-TI cDNA was isolated based on sequence information from purified GlcNAc-TI (Sarkar etal., 1991) and used to clone a portion of the human GlcNAc-TI gene (Hull et al., 1991). Recently, the coding region of a mouse GlcNAc-TI gene was isolated (Pownall etal, 1992). The GlcNAc-TI proteins from different species are unrelated to other glycosyltransferases, but like all mammalian glycosyltransferases cloned to date, are Type n transmembrane proteins in which the catalytic domain is in the C-terminal portion of the protein (Paulson and Colley, 1989; Lowe, 1991). In this paper, the sequence of a mouse GlcNAc-TI cDNA is presented. Subclones derived from the cDNA have been used to express active GlcNAc-TI in Escherichia coli, and to partially characterize the mouse GlcNAc-TI gene and its transcriptional products.

R.Kumar et al.

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Cloning/expression of mouse GlcNAc-TI gene

Bacterial expression of mouse GlcNAc-TI In order to show that the mouse cDNA in Figure 1 encodes active GlcNAc-TI enzyme, the C-terminal portion of the coding region was expressed in E. coli. Although the human GlcNAcTI gene has been expressed in transfected Lecl CHO cells (Kumar et al., 1990; Hull et al., 1991), and the rabbit GlcNAcTI cDNA was expressed by in vitro transcription and translation (Sarkar et al., 1991), the mouse gene isolated by Pownall et al. (1992) was not expressed and expression of GlcNAc-TI in bacteria has not been reported. To obtain bacterial expression, the Al GlcNAc-TI cDNA fragment (Figure 1) lacking information for the first 34 amino acids (i.e. the entire cytoplasmic and transmembrane regions) was cloned into the BamHl site of the S10 leader gene in the pET.3b vector to create pET.3b.GlcNAc-TI. Following transformation into E.coli strain BL21(DE3), which encodes T7 polymerase under the control of the LacUV5 promoter, inducible expression of the fusion gene in the vector is achieved by treatment with isopropyl-/3-r>thiogalactopyranoside (IPTG). Initial experiments compared the protein profile and GlcNAcTI activities of cell lysates from BL21(DE3) cells transformed with pET.3b.GlcNAc-TI or pET.3b containing the GlcNAc-TI gene in the opposite orientation (pET.3b.IT-cANclG). Lysates from BL21(DE3) cells carrying pET.3b.GlcNAc-TI exhibited a new protein band of — 45 kDa (the predicted molecular weight of the SlO-GlcNAc-TI fusion protein) and a GlcNAc-TI activity of > 120 nmol/h/mg protein in cell lysates after 1 h of IPTG induction. Untransformed cells or those transformed with anti-sense GlcNAc-TI had no extra protein band or GlcNAc-TI activity. However, uninduced cell lysates from BL21(D3)pET.3b.GlcNAc-TI transformants contained significant GlcNAc-TI activity (96 nmol/h/mg protein). This pre-induction activity was markedly reduced when the pET.3b.GlcNAc-TI plasmid was transformed into BL21(DE3) carrying pLysS (Table I) which expresses lysozyme and thereby inactivates low levels of T7 RNA polymerase that may derive from transcription of the T7 polymerase gene (Studier et al., 1990). The data in Figure 2 show the appearance of the - 45 kDa fusion protein in BL21(DE3)pLsyS cells and the corresponding GlcNAc-TI activities are given in Table I. The highest GlcNAc-TI activity was routinely obtained at 1 h post-induction. The decrease in enzyme activity, with time, despite the continuing increase in

Table I. GlcNAc-TI activity of bacteria] lysates Cell lysate

Induction with IPTG (h)

GlcNAc-TI activity (nmol/h/mg protein)

Untransformed host cells

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Cloning and expression of the murine gene and chromosomal location of the human gene encoding N-acetylglucosaminyltransferase I.

A mouse cDNA clone previously isolated from an F9 teratocarcinoma cell library and shown to confer N-acetylglucosaminyltransferase I (GlcNAc-TI) activ...
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