Analysis of the substrate binding sites of human galactosyltransferase by protein engineering.

Abstract

An expression vector, pIN‐GT, encoding the soluble form of beta 1,4‐galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN‐III‐ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN‐GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti‐GT antibodies. The recombinant GT was purified to homogeneity by N‐acetylglucosamine‐Sepharose affinity chromatography. The NH2‐terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP‐galactose analog, 4‐azido‐2‐nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site‐directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287––Phe remained as active as wild‐type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N‐acetyglucosamine binding and Tyr309 is involved in UDP‐galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Golgi Localization and in Vivo Activity of a Mammalian Glycosyltransferase (Human β1,4-Galactosyltransferase) in Yeast

“…If the Mnt1/Gal-Tf fusions had inserted into yeast organelle membranes as the authentic Mnt1 and Gal-Tf proteins (4,11,34), which are type II membrane proteins, it could be expected that they are refractory to the action of proteases. To test this notion crude extracts of phMGT1 and phMGT3 transformants were treated with proteinase K in the presence and absence of Triton X-100; subsequent to the treatments the Gal-Tf fusion protein was analyzed on immunoblots using the anti-Gal-Tf antibody.…”

“…The complex type of N-glycosyl chains in mammalian cells is synthesized after trimming of the core unit by the sequential addition of GlcNAc, Gal, and NeuAc. Galactose addition in human cells is mediated by UDP-galactose: Nacetyl-D-glucosaminyl-glycopeptide 4␤-D-galactosyltransferase (EC 2.4.1.38) (Gal-Tf), 1 an enzyme anchored in the Golgi membrane, which uses UDP-Gal as substrate and GlcNAc on glycosyl structures as acceptors (3,4). In its secreted soluble form, which lacks a membrane anchor region, Gal-Tf functions as lactose synthase, while a third role of Gal-Tf, correlated with its localization on the cell surface, is to mediate adhesion processes (5).…”

Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in Saccharomyces cerevisiae

“…Thus kinetic studies of mutagenized soluble hGal-T, expressed in different systems, have been extensively used to identify amino acids important for its catalytic activity. Thus it has been shown that Tyr284, Tyr309 and Trp310 are critically involved in GlcNAc binding and Tyr309 involved in UDP-Gal binding (Aoki et al, 1990). Also, based on kinetic studies on mutagenized enzyme forms, it has been proposed that Cys129 and Cys245 are implicated in a disulfide bond which holds the Gal-T in a conformation that is required for its catalytic activity and Cys340 may be involved in the binding of UDP-Gal (Wang et al, 1994).…”

A Mutagenic Study of β-1,4-Galactosyltransferases from Neisseria meningitidis

“…The availability of galactosyltransferase cDNA opens the way to the investigation of the structure-function relationships of galactosyltransferase by recombinant techniques. The functional domains of mammalian galactosyltransferase have been studied by various laboratories by expressing cDNA clones in both bacterial and mammalian cells (Masibay and Qasba, 1989;Aoki et al, 1990;Nakazana et al, 1993). Based on amino acid sequence similarities and conserved structural features, eucaryotic and bacterial galactosyltransferases can be classified into five families; A, B, C, D and E (Breton et al, 1998).…”

“…Region II contains an acidic motif, EDD, surrounded by two stretches of hydrophobic residues, which exhibits hydrophobic cluster analysis (Gaboriaud et al, 1987) similarity with the DVD and DXD motifs of families A and B, respectively (Breton et al, 1998). This is located in a region of the catalytic domain that has been implicated in UDPgalactose binding by chemical Brew, 1990, 1991) and mutational studies (Aoki et al, 1990;Zu et al, 1995) The structural and functional relationships of mammalian galactosyltransferase have been well studied, but bacterial galactosyltransferase is poorly understood. Recently, we cloned and expressed β-1,4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae (Park et al, 2002).…”