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Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs., Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

The gene B3GALT6 encodes for the UDP-Gal:βGalβ1-4Xyl β1,3-galactosyltransferase (β3Gal-T6) previously designated the Gal-II synthase catalyzing the third step in the biosynthesis of the proteoglycan linker tetrasaccharide (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser). The β1,3-galactosyltransferase family assigned to CAZy family GT31 contains 6 members, β3Gal-T1, T2, T4, T5, T6, and C1Gal-T1 with different functions. β3Gal-T1, -T2, and -T5 form Galβ1-3GlcNAc linkages (type 1 chain structures) in glycolipids and glycoproteins (Kolbinger et al. 1998; Amado et al. 1998; Hennet et al. 1998; Isshiki et al. 1999; Zhou et al. 1999a), β3Gal-T4 forms Galβ1-3GalNAc in gangliosides (Amado et al. 1998), β3Gal-T6 forms Galβ1-3Gal in the proteoglycan linker region (Bai et al. 2001), and C1Gal-T1 forms Galβ1-3GalNAca1-O-Ser/Thr (core 1 structure) in O-glycans (Ju et al. 2002). The acceptor molecules for these enzymes include glycosphingolipids, N-glycoproteins, mucin-type O-glycoproteins, and proteoglycans (Miyazaki et al. 1997; Amado et al. 1999).

The Galβ1-3GlcNAc linkage (type 1 chain) is widely found on glycolipids and N-linked and mucin-type O-linked glycoproteins primarily in cell types derived from the endoderm (Oriol et al. 1986). In contrast, the Galβ1-4GlcNAc linkage (type 2) is widely distributed on glycolipids and glycoproteins derived from the ectoderm and mesoderm. Type 1 chain constitutes cores for the Lewis blood group antigens including structures such as Lea and Leb and the corresponding ABH structures (Clausen and Hakomori 1989). Type 1 chain also carries classical cancer-associated structures such as sialyl-Lea (N19-9) (Magnani et al. 1982) and disialyl-Lea (FH7) (Nudelman et al. 1986). The type 1 chain core is catalyzed by β3galactosyltransferases (β3Gal-T) and distinct β3Gal-T activities with βGlcNAc substrates were first described in the 1980s (Sheares and Carlson 1983; Sheares et al. 1982) and shown to be distinct from β4galactosyltransferase (β4Gal-T) activities (Holmes 1989; Sheares and Carlson 1983). The first β3Gal-T gene cloned, β3Gal-T1, was identified by expression cloning (Sasaki et al. Japanese patent JP1994181759-A/1). Subsequently, this gene sequence was used to probe emerging EST and gene sequences early in the human genome project and unravel the large β3glycosyltransferase gene family assigned to CAZy family GT31. This family includes three β3Gal-Ts (T1, T2, and T5) catalyzing the Galβ1-3GlcNAc linkage as well as a large number of other β3GalNAc-Ts, β3GlcNAc-Ts (for reviews see Amado et al. 1999; Togayachi et al. 2006). This review summarizes the properties of the first two cloned and expressed β3Gal-Ts, β3Gal-T1 and T2.

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.

Complex carbohydrates serve a wide range of biological functions in cells and tissues. Their biosynthesis involves more than 200 distinct glycosyltransferases in human cells, and the expression, properties, and topology of these enzymes regulate the glycosylation patterns of proteins and lipids. Glycosyltransferases are ER-Golgi resident enzymes with slow turnover, which makes monitoring of protein expression a method more directly linked to enzyme function, than monitoring gene expression. In situ monitoring of expression and subcellular topology of glycosyltransferase proteins by immunological techniques using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating and characterizing monoclonal antibodies to human glycosyltransferases. This strategy includes a process for recombinant production and purification of enzymes for immunization, a simple selection strategy for isolation of antibodies with optimal properties for in situ detection of enzyme expression, and a comprehensive strategy for characterizing the fine specificity of such antibodies.

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His6. At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5–10nM LD. LDI retained stability in the pH 2–12 range and at pH 6.5 displayed a half-life of 53 and 33min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K(m,app)=0.16+/-0.02 mg/mL and k(cat,app)=79+/-10s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K(m,app)=488+/-23mL/(mgs) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K(d) of 27.2, 0.70, and 34.7 microM, respectively.