Fucose Incorporation Research Articles

Incorporation of fucose into glycans independent of the GDP-fucose transporter SLC35C1 preferentially utilizes salvaged over de novo GDP-fucose

Mutations in the SLC35C1 gene encoding the Golgi GDP-fucose transporter are known to cause leukocyte adhesion deficiency II. However, improvement of fucosylation in leukocyte adhesion deficiency II patients treated with exogenous fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which remains a mystery. To investigate this phenomenon, we developed and characterized a human cell–based model deficient in SLC35C1 activity. The resulting cells were cultured in the presence/absence of exogenous fucose and mannose, followed by examination of fucosylation potential and nucleotide sugar levels. We found that cells displayed low but detectable levels of fucosylation in the absence of SLC35C1. Strikingly, we show that defects in fucosylation were almost completely reversed upon treatment with millimolar concentrations of fucose. Furthermore, we show that even if fucose was supplemented at nanomolar concentrations, it was still incorporated into glycans by these knockout cells. We also found that the SLC35C1-independent transport preferentially utilized GDP-fucose from the salvage pathway over the de novo biogenesis pathway as a source of this substrate. Taken together, our results imply that the Golgi systems of GDP-fucose transport discriminate between substrate pools obtained from different metabolic pathways, which suggests a functional connection between nucleotide sugar transporters and nucleotide sugar synthases.

Fluorescent glycan fingerprinting of SARS2 spike proteins

Glycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.

Detecting substrate glycans of fucosyltransferases with fluorophore-conjugated fucose and methods for glycan electrophoresis.

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and 3H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

Applying Acylated Fucose Analogues to Metabolic Glycoengineering.

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody’s binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering.

Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells

Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFR-mediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.

A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

The Bacteroides are a numerically dominant genus of the human intestinal microbiota. These organisms harbor a rare bacterial pathway for incorporation of exogenous fucose into capsular polysaccharides and glycoproteins. The infrequency of glycoprotein synthesis by bacteria prompted a more detailed analysis of this process. Here, we demonstrate that Bacteroides fragilis has a general O-glycosylation system. The proteins targeted for glycosylation include those predicted to be involved in protein folding, protein-protein interactions, peptide degradation as well as surface lipoproteins. Protein glycosylation is central to the physiology of B. fragilis and is necessary for the organism to competitively colonize the mammalian intestine. We provide evidence that general O-glycosylation systems are conserved among intestinal Bacteroides species and likely contribute to the predominance of Bacteroides in the human intestine.

Characterization and role of fucose mutarotase in mammalian cells

L-Fucose for mammalian glycosylation contains an anomeric carbon atom generating alpha- and beta-L-fucoses. Based on sequence comparison of mouse and human homologs with the prokaryotic fucose mutarotases (FucU) characterized previously, we investigated their function in mammalian cells. By nuclear magnetic resonance (NMR) measurement with saturation difference analysis, the purified mammalian mutarotases were demonstrated to be involved in an interconversion between the two anomeric forms with comparable efficiency as that of the Escherichia coli FucU. The mouse gene was widely expressed in various tissues and cell lines, including kidney, liver, and pancreas, although expression was marginal in muscle and testis. By generating stably expressed cell lines for mutarotase genes in HepG2, it was shown that fucose incorporations into cellular proteins were increased as demonstrated by an incorporation of radiolabeled fucose into the cells. Furthermore, intracellular levels of GDP-L-fucose, measured with high performance liquid chromatography (HPLC), were enhanced by an overproduction of cellular mutarotase, which was reversed by gene silencing of mutarotase based on RNA interference. The results suggest that the mammalian mutarotase is functional in facilitated incorporation of fucose through the salvage pathway.

Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts

Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (alpha1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of alpha1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.

Quantitative Trait Loci Associated with Cell Wall Polysaccharides in Soybean Seed

Seed cell wall polysaccharides (CWPs) represent a significant portion of seed dry matter (DM) in soybean [Glycine max (L.) Merr.]. Quantitative trait locus (QTL) analysis is a step toward identifying genes controlling CWP concentration and composition of soybean seed. The objectives of this study were to identify QTLs associated with CWP variability and investigate the relationships between seed CWP, protein, and oil content. Whole soybean seed from 'Minsoy', 'Archer', and 108 Minsoy × Archer recombinant inbred (RI) lines were analyzed for CWP using the Uppsala total dietary fiber method. For a random subsample of 73 RI lines, embryos (seed with the seed coats removed) were analyzed. Three QTLs were observed on Linkage Groups U3 (ISU A2), U7 (ISU Al), and U24 (ISU K) that represented most of the variability in CWP content expressed on a DM basis in both whole seed and embryos. A QTL for fucose was detected on U3. Linkage Group U7 contained multiple QTLs mapping at the same location for galactose, the arabinose-to-galactose ratio (Ara/ Gal), pectin, and CWP content. A QTL for arabinose was on U24. These same chromosomal regions also exhibited significant QTLs when the monosaccharide data were expressed on a CWP basis, suggesting that most of the variation in CWP was controlled by changes in the incorporation of galactose, arabinose, and fucose into CWP as total CWP concentration increased. Negative correlations of protein with oil, and the sum of protein and oil with most monosaccharides, pectin, and CWP were present within the RI population, indicating that decreasing seed CWP content will improve seed quality.

Differential Terminal Fucosylation of N-Linked Glycans Versus Protein O-Fucosylation in Leukocyte Adhesion Deficiency Type II (CDG IIc)

LAD II/CDG IIc is a rare autosomal recessive disease characterized by a decreased expression of fucosylated antigens on cell surfaces that results in leukocyte adhesion deficiency and severe neurological and developmental abnormalities. Its molecular basis has been identified as a defect in the transporter of GDP-l-fucose into the Golgi lumen, which reduces the availability of the substrate for fucosyltransferases. During metabolic radiolabeling experiments using [3H]fucose, LAD II fibroblasts incorporated significantly less radiolabel compared with control cells. However, fractionation and analysis of the different classes of glycans indicated that the decrease in [3H]fucose incorporation is not generalized and is mainly confined to terminal fucosylation of N-linked oligosaccharides. In contrast, the total levels of protein O-fucosylation, including that observed in Notch protein, were unaffected. This finding demonstrates that the decrease in GDP-l-fucose levels in the fibroblast Golgi caused by the LAD II defect does not impair bulk protein O-fucosylation, but severely affects the bulk addition of fucose as a terminal modification of N-linked glycans. These data suggest that the severe clinical abnormalities including neurological and developmental ones observed in at least some of the LAD II patients may be related to alteration in recognition systems involving terminal fucose modifications of N-glycans and not be due to a defective O-fucosylation of proteins such as Notch.

Fucosyltransferases in Schistosoma mansoni development.

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.

Isolation and characterization of [ 3H]fucose-labeled glycoproteins from the serum of normal rats and rats bearing Zajdela hepatoma

Specific [ 3H]fucose-labeled glycoproteins were found in the serum of rats bearing Zajdela hepatoma, which possess different isoelectric points in comparison with serum glycoproteins from normal rats. The electrophoretic profile of the serum glycoproteins is significantly altered. There is approximately a 2.5 times increase of [ 3H]fucose incorporation into serum glycoproteins from rats with an ascitic form of hepatoma, compared with normal rats and to animals bearing a solid form of the tumor (which have a considerably greater survival). Serum fucoproteins identical for the two forms of hepatoma were isolated, as well as glycoproteins strongly specific for each of the forms.

Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium.

Expression of the beta-galactoside alpha 1,2-fucosyltransferase gene suppresses axonal outgrowth of neuro2a neuroblastoma cells.

The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the beta-galactoside alpha 1,2-fucosyltransferase (alpha 1,2-FT) gene. We recently cloned two types of rabbit alpha 1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene alpha 1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene alpha 1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and alpha-L-fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H-type alpha1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.

Glycosidic specificity of fucosyltransferases present in rat epididymal spermatozoa.

We have recently demonstrated multiple fucosyltransferase (FT) activity in rat spermatogenic cells. To complement these findings, here we identify and partially characterize the glycosidic linkage specificity of FTs present in spermatozoa from caput and cauda epididymides. Analysis of the acceptor substrate specificity of the FTs by thin-layer chromatography indicated that both caput and cauda sperm expressed alpha(1-2)-, alpha(1-3)-, alpha(1-4)-FTs as demonstrated by fucose incorporation into phenyl-beta-D-galactoside, 2'-fucosyllactose, and lacto-N-fucopentaose-I, respectively. Spermatozoa from the cauda epididymidis exhibited significant decreases in the levels of alpha(1-2)-, alpha(1-3)-, alpha(1-4)-FTs, and of total soluble FTs in comparison to spermatozoa from the caput epididymidis. The relative ratio of alpha(1-3)-FT to total FT activity appeared to be significantly higher than those of alpha(1-2)- or alpha(1-4)-FTs, in spermatozoa both from caput and cauda epididymides. Using different types of low molecular weight acceptors and the selective inhibition of the FT by N- ethylmaleimide, we have demonstrated that at least alpha(1-2)-FT is different from alpha(1-3)- or alpha(1-4)-FTs. Kinetic studies also showed that alpha(1-2)-FT is different from alpha(1-3)- or alpha(1-4)-FTs as demonstrated by apparent Km and Vmax values. Moreover, alpha(1-3)- and alpha(1-4)-FT activities in cauda sperm were found to be highly sensitive to Mn2+ but showed differential responses to divalent cations. In contrast, both alpha(1-3)- and alpha(1-4)-FTs seemed to be relatively less sensitive to Mg2+. Thus, these results not only demonstrate the presence of multiple FTs in rat epididymal sperm but also differentiate individual FTs with regard to their kinetic properties and sensitivity to both inhibitor and divalent cations.

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewis(a) secretion in colorectal carcinoma cultures.

The rate of polarised secretion of sialosyl Lewis(a)(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 microM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis(a) levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis(a) epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 M(r) on acrylamide gradient gels were concentrated in two doublets in the apparent M(r) range 106,000-152,000 on Western blots. Carbohydrate analyses of oligosaccharides from SiaLeams of membrane subfractions and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis(a), fucose/total CHO, and (3H) fucose incorporation in control samples than RA samples. Western blots of samples from membrane subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis(a) epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis(a) induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Gal beta 1-3 moieties at oligosaccharide termini destined for export from the Golgi.

Presence of multiple fucosyltransferases in rat Sertoli cells and spermatogenic cells.

Differential expression of fucosyltransferases (FTs) on Sertoli cell and germ cell surfaces and their function as ectoenzymes may be important in the process of spermatogenesis. To determine the glycosidic linkage specificity of FTs present in cultured Sertoli cells and in germ cells, we quantified FT activities by thin-layer chromatography using both high and low molecular weight acceptors in the presence of GDP-[14C]-L-fucose. Analysis of the acceptor substrate specificity of the FTs indicated that alpha(1-2), alpha(1-3), alpha(1-4)-FTs are expressed as demonstrated by fucose incorporation into phenyl-beta-D-galactoside, 2'-fucosyllactose, and lacto-N-fucopentaose-I, respectively. In Sertoli cells, the ratios of the three FTs examined were the same for whole-cell extracts and samples of purified plasma membranes. Higher relative FT activity was observed in plasma membranes from mixed germ cells than in Sertoli cell membranes. Furthermore, alpha(1-3)-FT and alpha(1-4)-FT activities were higher in mixed germ cell membranes. Spermatogenic stage specificity of FT expression was assessed in purified populations of germ cells. With calculation on a per-cell basis, all three alpha-FTs exhibited a quantitative decrease during the transition between pachytene spermatocytes and round spermatids. The decrease in alpha(1-3)-FT activity was particularly significant. In rat germ cells, all three alpha-FT activities associated with the cell surface in pachytene spermatocytes and round spermatids were 34-53% and 52-53%, respectively, of the total cell FT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Putrescine on Tissue Transglutaminase Activity in Wounds

Topical application of putrescine, a transglutaminase inhibitor, for 3 days directly to rat skin wounds produced a significant average decrease of 48 percent in wound breaking strength in test animals from 8 pairs studied between day 5 and day 10 after wounding. No external or systemic toxic effects of putrescine were seen with localized topical application of 50 mM putrescine for 3 days in any of the test rats (n = 12), and no systemic toxicity was seen in rabbits (n = 4) after topical exposure to 50 mM putrescine for 3 weeks. Quantitation of tritiated fucose incorporation in rat wound explants from 10 pairs of rats revealed that a significant overall decrease in radiolabeled glycoprotein production of 23 percent occurred when putrescine was present; in addition, the fraction of tritiated glycoprotein which was soluble in buffer was significantly increased, while that in the buffer-insoluble fraction decreased. This study suggests that putrescine inhibits tissue transglutaminase-mediated cross-linking of fucoprotein in the extracellular wound matrix and supports a role for this process in the generation of incisional wound strength.

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans.

The prestalk and prespore cells from the Dictyostelium discoideum multicellular slug stage of development differ in assembly of glycoconjugates. Prespore cells are 2- to 3-fold more active than prestalk cells in the assembly of N-linked glycans and 20-fold more active in their fucosylation. Only prespore cells synthesize an O-linked glycan consisting in part of Fuc alpha-linked to N-acetylglucosamine. Incorporation of fucose, glucosamine, mannose and galactose into large pronase-resistant glycoconjugates was almost exclusively into prespore cells. Such glucosamine-labelled glycoconjugates resist fragmentation by beta-elimination and include a glycoantigen dependent on the modB genetic locus. In contrast, large fucose-labelled glycoconjugates consisted of multiple, small, O-linked oligosaccharides on carrier peptides. The spore coat protein SP96 has several fucosylated O-linked oligosaccharides, one of which correlates with a fucose epitope previously shown to localize in prespore vesicles and the outer layer of the spore coat.