Apoptosis and involution in the mammary gland are altered in mice lacking a novel receptor, beta1,4-Galactosyltransferase I

Receptor-mediated cell-extracellular matrix (ECM) interactions are critical regulators of cell survival, and perturbing these signaling pathways can disrupt cellular differentiation and function in a variety of tissues, including the mammary gland. One such receptor is the cell surface-associated, long isoform of beta1,4-galactosyltransferase I (GalT I). Deletion of long GalT I leads to increased mammary ductal branching morphogenesis [Dev. Biol., 244 (2002) 114]. Here, we show that this expansion in the mammary epithelial (ME) cell compartment is accomplished through decreased apoptosis during pregnancy and involution. Decreased apoptosis during involution is concomitant with delayed alveolar collapse, persistent expression of the milk protein gene alpha-lactalbumin and delayed expression of genes associated with the tissue-remodeling phase of involution. Using 3-dimensional in vitro cultures, we show that the decrease in apoptosis is dependent on laminin 1, a ligand for surface GalT I, suggesting that surface GalT I negatively influences ECM-dependent cell survival, a novel function for an ECM receptor. In the best-studied examples, ECM promotes survival through integrin receptor-mediated activation of focal adhesion kinase (FAK). Aggregation of surface GalT I also activates FAK, therefore, we asked if FAK activation was altered in ME from long GalT I null mice. Activated FAK was appropriately localized to focal adhesions in long GalT I null ME. However, FAK activation was constitutively reduced 4.5-fold in long GalT I nulls relative to wild type. Expression of the integrin beta1 subunit was not affected by loss of long GalT I. Collectively, these results suggest that surface GalT I might negatively regulate ME cell survival by linking integrin-independent FAK activation to apoptotic rather than survival signaling events.     

Mice Overexpressing β-1,4-Galactosyltransferase I Are Resistant to TNF-Induced Inflammation and DSS-Induced Colitis

Glycosylation is an essential post-translational modification, which determines the function of proteins and important processes such as inflammation. β-1,4-galactosyltransferase I (βGalT1) is a key enzyme involved in the addition of galactose moieties to glycoproteins. Intestinal mucins are glycoproteins that protect the gut barrier against invading pathogens and determine the composition of the intestinal microbiota. Proper glycosylation of mucus is important in this regard. By using ubiquitously expressing βGalT1 transgenic mice, we found that this enzyme led to strong galactosylation of mucus proteins, isolated from the gut of mice. This galactosylation was associated with a drastic change in composition of gut microbiota, as TG mice had a significantly higher Firmicutes to Bacteroidetes ratio. TG mice were strongly protected against TNF-induced systemic inflammation and lethality. Moreover, βGalT1 transgenic mice were protected in a model of DSS-induced colitis, at the level of clinical score, loss of body weight, colon length and gut permeability. These studies put βGalT1 forward as an essential protective player in exacerbated intestinal inflammation. Optimal galactosylation of N-glycans of mucus proteins, determining the bacterial composition of the gut, is a likely mechanism of this function.     

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP- galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.      

Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.     

Expression of natural human β1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans

β1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human β1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.     

Localization of Triton-insoluble cAMP-dependent kinase type RIβ in rat and mouse brain

In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RIα and β, RIIα and β). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RIβ isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RIα and RIβ isoforms is bound to the cytoskeleton. RIβ labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RIα aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RIα isoforms, while the RIβ isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RIα and β isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions.     

Expression of β‐1,4‐galactosyltransferase I in rat Schwann cells

Glycosylation is one of the most important post‐translational modifications. It is clear that the single step of β‐1,4‐galactosylation is performed by a family of β‐1,4‐galactosyltransferases (β‐1,4‐GalTs), and that each member of this family may play a distinct role in different tissues and cells. In the present study, real‐time PCR revealed that the β‐1,4‐GalT I mRNA reached peaks at 2 weeks after sciatic nerve crush and 3 days after sciatic nerve transection. Combined in situ hybridization for β‐1,4‐GalT I mRNA and immunohistochemistry for S100 showed that β‐1,4‐GalT I mRNAs were mainly located in Schwann cells after sciatic nerve injury. In conclusion, β‐1,4‐GalT I might play important roles in Schwann cells during the regeneration and degeneration of the injured sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affected β‐1,4‐GalT I mRNA expression in sciatic nerve in a time‐ and dose‐dependent manner, and β‐1,4‐GalT I mRNA is expressed mainly in Schwann cells. These results indicated that β‐1,4‐GalT I plays an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that β‐1,4‐GalT I in Schwann cells in vitro was affected in a time‐ and concentration‐dependent manner in response to LPS stimulation. All these results suggest that β‐1,4‐GalT I play an important role in Schwann cells in vivo and vitro during pathology. In addition, β‐1,4‐GalT I production was drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that Schwann cells which regulated β‐1,4‐GalT I expression after LPS stimulation were via ERK, SAPK/JNK, and P38 MAP kinase signal pathways. J. Cell. Biochem. 108: 75–86, 2009. © 2009 Wiley‐Liss, Inc.     

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.     

Differential arginylation of actin isoforms: the mystery of the actin N-terminus

Actin is one of the most abundant, essential and well studied intracellular proteins, yet its regulation in vivo is still not completely understood. One of the mysteries around actin concerns the existence of multiple actin isoforms that are extremely similar to each other except for their N-termini but have been shown in multiple studies to preferentially incorporate into different actin networks and are suggested to have different roles in vivo. The mechanisms of this actin isoform segregation are unknown. My colleagues and I recently showed that beta but not gamma actin in cultured fibroblasts undergoes N-terminal arginylation, which regulates actin polymerization and lamella formation in motile cells. Here, I propose that arginylation could be a general mechanism that regulates actin isoform segregation in vivo and participates in the formation of loose beta-actin network at the leading edge of the cell.     

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.     

The secreted immunoglobulin domain proteins ZIG-5 and ZIG-8 cooperate with L1CAM/SAX-7 to maintain nervous system integrity

During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig) domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L) and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S). Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins.     

Sperm from beta1,4-galactosyltransferase I-null mice exhibit precocious capacitation

Mammalian sperm must undergo a physiological maturation, termed capacitation, before they are able to fertilize eggs. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. In this paper, we describe the capacitation phenotype of sperm lacking the long isoform of beta1,4-galactosyltransferase I (GalT I), a sperm surface protein that functions as a receptor for the zona pellucida glycoprotein, ZP3, and as an inducer of the acrosome reaction following ZP3-dependent aggregation. As expected, wild-type sperm must undergo capacitation in order to bind the zona pellucida and undergo a Ca(2+) ionophore-induced acrosome reaction. By contrast, GalT I-null sperm behave as though they are precociously capacitated, in that they demonstrate maximal binding to the zona pellucida and greatly increased sensitivity to ionophore-induced acrosome reactions without undergoing capacitation in vitro. The loss of GalT I from sperm results in an inability to bind epididymal glycoconjugates that normally maintain sperm in an 'uncapacitated' state; removing these decapacitating factors from wild-type sperm phenocopies the capacitation behavior of GalT I-null sperm. Interestingly, capacitation of GalT I-null sperm is independent of the presence of albumin, Ca(2+) and HCO(3)(-); three co-factors normally required by wild-type sperm to achieve capacitation. This implies that intracellular targets of albumin, Ca(2+) and/or HCO(3)(-) may be constitutively active in GalT I-null sperm. Consistent with this, GalT I-null sperm have increased levels of cAMP that correlate closely with both the accelerated kinetics and co-factor-independence of GalT I-null sperm capacitation. By contrast, the kinetics of protein tyrosine phosphorylation and sperm motility are unaltered in mutant sperm relative to wild-type. These data suggest that GalT I may function as a negative regulator of capacitation in the sperm head by suppressing intracellular signaling pathways that promote this process.     

The 14-3-3 proteins of Trypanosoma brucei function in motility, cytokinesis, and cell cycle

The cDNAs for two isoforms (I and II) of the 14-3-3 proteins have been cloned and functionally characterized in Trypanosoma brucei. The amino acid sequences of isoforms I and II have 47 and 50% identity to the human tau isoform, respectively, with important conserved features including a potential amphipathic groove for the binding of phosphoserine/phosphothreonine-containing motifs and a nuclear export signal-like domain. Both isoforms are abundantly expressed at approximately equal levels (1-2 x 10(6) molecules/cell) and localized mainly in the cytoplasm. Knockdown by induction of double-stranded RNA of isoform I and/or II in both bloodstream and procyclic forms resulted first in a reduction of cell motility and then significant reduction in cell growth rates and morphological changes; the changes include aberrant numbers of organelles and abnormal shapes and sizes that mimic phenotypes produced by various cytokinesis inhibitors. Morphological and fluorescence-activated cell sorting analysis of the cell cycle suggested that isoforms I and II might play important roles in nuclear (G2-M transition) and cell (M-G1 transition) division. These findings indicate that the 14-3-3 proteins play important roles in cell motility, cytokinesis, and the cell cycle.     

Differential recognition of Toxoplasma gondii recombinant nucleoside triphosphate hydrolase isoforms by naturally infected human sera

Toxoplasma gondii possesses a highly active nucleoside triphosphate hydrolase, which has been shown to be an immunodominant antigen in mice and humans. Two isoforms (I and II) which exhibit different activities with respect to hydrolysis of ATP exist. Past studies suggest that all strains of T. gondii contain the less active nucleoside triphosphate hydrolase II, whilst only virulent strains contain the nucleoside triphosphate hydrolase I isoform. In order to further investigate the correlation between nucleoside triphosphate hydrolase isoform and biological significance, we cloned and expressed as glutathione S-transferase fusion proteins the full-length nucleoside triphosphate hydrolase I and II isoforms and two truncations of the nucleoside triphosphate hydrolase I isoform in Escherichia coli. We then used ELISAs with the full-length recombinant nucleoside triphosphate hydrolases as antigens to examine 188 naturally infected T. gondii-positive sera and 83 T. gondii-negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite lysate antigen, 31 sera reacted to both nucleoside triphosphate hydrolase isoforms, three sera reacted specifically to nucleoside triphosphate hydrolase I and two sera reacted to only nucleoside triphosphate hydrolase II. Immunoblot analysis of the five sera reacting to either nucleoside triphosphate hydrolase I or II revealed both quantitative and qualitative differences in reactivity to the two isoforms. Comparative immunoblot analysis using the truncations of the nucleoside triphosphate hydrolase I isoform, and one of these positive sera identified a presumptive differential epitope between the nucleoside triphosphate hydrolase I and II isoforms within an 81-aa region (aa 445–526) at the C-terminus of the nucleoside triphosphate hydrolase I isoform. This differential reactivity was further localised to the 12-residue region of greatest variability between the two isoforms (residues 488–499) using synthetic peptides. This is the first report where naturally infected human sera have been used to identify a differential epitope. Because this region is essential for substrate binding, an antibody response to this region may play some role in inhibition of this highly active enzyme.     

Functional identification of galactosyltransferases (SCGs) required for species-specific modifications of the lipophosphoglycan adhesin controlling Leishmania major-sand fly interactions

Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Galbeta1,4Manalpha1-PO(4)) repeating units. In L. major these are extensively substituted with Gal(beta1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain beta1,3-galactosyltransferases (betaGalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain betaGalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression.