Nadia Malagolini

Tamm-Horsfall glycoprotein: biology and clinical relevance

Tamm-Horsfall glycoprotein (THP) is the most abundant urinary protein in mammals. Urinary excretion occurs by proteolytic cleavage of the large ectodomain of the glycosyl phosphatidylinositol-anchored counterpart exposed at the luminal cell surface of the thick ascending limb of Henles loop. We describe the physical-chemical structure of human THP and its biosynthesis and interaction with other proteins and leukocytes. The clinical relevance of THP reported here includes: (1) involvement in the pathogenesis of cast nephropathy, urolithiasis, and tubulointerstitial nephritis; (2) abnormalities in urinary excretion in renal diseases; and (3) the recent finding that familial juvenile hyperuricemic nephropathy and autosomal dominant medullary cystic kidney disease 2 arise from mutations of the THP gene. We critically examine the literature on the physiological role and mechanism(s) that promote urinary excretion of THP. Some lines of research deal with the in vitro immunoregulatory activity of THP, termed uromodulin when isolated from urine of pregnant women. However, an immunoregulatory function in vivo has not yet been established. In the most recent literature, there is renewed interest in the capacity of urinary THP to compete efficiently with urothelial cell receptors, such as uroplakins, in adhering to type 1 fimbriated Escherichia coli. This property supports the notion that abundant THP excretion in urine is promoted in the host by selective pressure to obtain an efficient defense against urinary tract infections caused by uropathogenic bacteria.

Mechanisms of cancer-associated glycosylation changes.

Cell membrane glycoconjugates undergo characteristic changes as a consequence of neoplastic transformation. The cancer-associated carbohydrate structures play key roles in cancer progression by altering the cell-cell and cell-environment interactions. In this review, we will discuss some of the most relevant cancer-associated carbohydrate structures, including the β1,6-branching of N-linked chains, the sialyl Lewis antigens, the α2,6-sialylated lactosamine, the Thomsen-Friedenreich-related antigens and gangliosides. We will describe the mechanisms leading to the expression of these structures and their interactions with sugar binding molecules, such as selectins and galectins. Finally, we will discuss how the glycosylation machinery of the cell is controlled by signal transduction pathways, epigenetic mechanisms and responds to hypoxia.

Sialosignaling: sialyltransferases as engines of self-fueling loops in cancer progression.

BACKGROUND Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression. SCOPE OF THE REVIEW To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression. MAJOR CONCLUSIONS We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen-Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information. GENERAL SIGNIFICANCE Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.

High-mannose oligosaccharides from human Tamm-Horsfall glycoprotein

The present communication reports the occurrence of high-mannose oligosaccharides on Tamm-Horsfall glycoprotein prepared from human pooled urine. The Pronase digest of the glycoprotein was fractionated by gel filtration and a high-mannose glycopeptide species was separated from complex-type glycopeptides. When high-mannose glycopeptides were digested with endo-β-N-acetylglucosaminidase H, followed by reduction with [3H]KBH4, three oligosaccharides were resolved by thin-layer chromatography. On the basis of chromatographic mobility and exoglycosidase digestions the composition Man7-, Man6-, and Man5-GlcNAc was assigned to the three oligosaccharides. Man6GlcNAc is by far the major component.

Rapid isolation of Tamm-Horsfall glycoprotein (uromodulin) from human urine

An isolation method for Tamm-Horsfall protein is described which is based on the observation that a diatomaceous earth filter is able to retain most of the glycoprotein present in urine and that the glycoprotein is easily desorbed from the filter by deionized water. This behaviour depends on the tendency of Tamm-Horsfall glycoprotein at normal urinary concentrations to form a gel in a solution containing mono- and divalent ions. By means of two-step filtration, the glycoprotein was purified to homogeneity. The yield was of about 20 mg/l of urine, and the time required for the isolation was approximately 5-6 h. This procedure should be particularly useful for preparing large amounts of Tamm-Horsfall glycoprotein oligosaccharides in order to investigate their potential use as immunosuppressive agents both in vitro and in vivo.

Differentiation -dependent expression of human beta-galactoside alpha 2,6-sialyltransferase mRNA in colon carcinoma CaCo-2 cells.

We have previously documented a dramatic elevation in the activity of α2,6-sialyltransferase towards Galβ1,4GlcNAc (EC 2.4.99.1) (α2,6ST) in CaCo-2 cells maintained in culture for several days after confluence to elicit a high degree of enterocytic differentiation phenotype. Northern analysis performed with a probe complementary to a region of human α2,6ST mRNA common to all known transcripts demonstrated that the expression of α2,6ST mRNA in CaCo-2 cells increased with the degree of differentiation. When probes complementary to 5′-untranslated exons (Y+Z or X) previously identified in transcripts isolated from human placenta and from several human lymphoblastoid cell lines were used, no hybridization signal with mRNA of CaCo-2 cells was found, as reported for the mRNA of hepatoma cell line HepG2 (Wang XC, Vertino A, Eddy RL, Byers MG, Jani-Sait SN, Shows TB, Lau JTY (1993)J Biol Chem268: 4355–61). These results support the notion that the major α2,6ST transcript of CaCo-2 cells was the hepatoma isoform or a new one, so far unreported. Consistent with the differentiation-dependent increase in α2,6ST-mRNA expression, an elevation of the reactivity withSambucus nigra agglutinin of differentiated CaCo-2 cell-surface was observed, indicating an enhanced α2,6-sialylation of membrane glycoconjugates.

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.

The biosynthesis of the selectin-ligand sialyl Lewis x in colorectal cancer tissues is regulated by fucosyltransferase VI and can be inhibited by an RNA interference-based approach

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3sialyllactosamine at a low concentration (Fuc-T(SLN)); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI>Fuc-TIII>Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T(SLN) activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T(SLN) activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.

The expression of soluble and cell-bound α2,6 sialyltransferase in human colonic carcinoma CaCo-2 cells correlates with the degree of enterocytic differentiation

alpha 2,6 sialyltransferase towards the N-acetyllactosaminyl sequence (alpha 2,6 ST, E.C. 2.4.99.1) is one of the major sialyltransferases in human colonic cells; it strongly increases in human colorectal tumors and is largely expressed in fetal and neonatal rat colon. In this study we demonstrate that human colon carcinoma CaCo-2 cells, which differentiate spontaneously into enterocytes when maintained confluent for several days, exhibit a very high expression of alpha 2,6 ST both in the cell-bound and soluble form. When the CaCo-2 cells were cultured on porous membranes the soluble alpha 2,6 ST was mainly detected in the medium collected from the chamber corresponding to the basolateral face of the monolayer. The soluble alpha 2,6 ST could be concentrated and purified from the alpha 2,3 sialyltransferase by affinity chromatography on Blue Sepharose.