https://www.ajkd.org/article/S0272-6386(03)00829-1/pdf

Tamm-Horsfall glycoprotein: biology and clinical relevance

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.

/pdf/human-plasma-protein-n-glycosylation-4aipovmwnb.pdf

Human plasma protein N-glycosylation

Glycoproteins in animal cells contain a variety of glycan structures that are added co- and/or posttranslationally to proteins. Of over 20 different types of sugar-amino acid linkages known, the two major types are N-glycans (Asn-linked) and O-glycans (Ser/Thr-linked). An abnormal mucin-type O-glycan whose expression is associated with cancer and several human disorders is the Tn antigen. It has a relatively simple structure composed of N-acetyl-D-galactosamine with a glycosidic α linkage to serine/threonine residues in glycoproteins (GalNAcα1-O-Ser/Thr), and was one of the first glycoconjugates to be chemically synthesized. The Tn antigen is normally modified by a specific galactosyltransferase (T-synthase) in the Golgi apparatus of cells. Expression of active T-synthase is uniquely dependent on the molecular chaperone Cosmc, which is encoded by a gene on the X chromosome. Expression of the Tn antigen can arise as a consequence of mutations in the genes for T-synthase or Cosmc, or genes affecting other steps of O-glycosylation pathways. Because of the association of the Tn antigen with disease, there is much interest in the development of Tn-based vaccines and other therapeutic approaches based on Tn expression.

/pdf/the-tn-antigen-structural-simplicity-and-biological-1s57unkh4v.pdf

The Tn Antigen—Structural Simplicity and Biological Complexity

Tamm-Horsfall Protein Binds to Type 1 Fimbriated Escherichia coli and Prevents E. coli from Binding to Uroplakin Ia and Ib Receptors

https://www.kidney-international.org/article/S0085-2538(15)50168-3/pdf

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation.

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz 1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4S(?1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2 to Man8GlcNAc2, in addition to a small amount of complex-type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.

/pdf/glycosylation-sites-and-site-specific-glycosylation-in-human-1ey84weq9i.pdf

Glycosylation sites and site-specific glycosylation in human Tamm-Horsfall glycoprotein

The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HPLC on LiChrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA-1. Characterizations were performed using 500-MHz and 600-MHz 1H-NMR spectroscopy, in combination with sialidase treatments. The type of characterized N-glycans ranged from monosulfated to trisulfated N-glycans, whereby the sulfate groups were present as 3-O-sulfated Gal (Gal3S) and 4-O-sulfated GalNAc (GalNAc4S). A compilation of the established structures is shown below. [structure in text]

/pdf/sulfated-di-tri-and-tetraantennary-n-glycans-in-human-tamm-y8i6ax0kgr.pdf

Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein.

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramnios patient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anionexchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chromatography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz 1 H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (α1‐3)-fucosylated N-glycans were found, representing sialyl Le x elements. Furthermore, as compared to hST, a higher degree of ( α1‐6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.

/pdf/expression-of-n-linked-sialyl-lex-determinants-and-o-glycans-1veag9zxti.pdf

Expression of N-linked sialyl Lex determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

containing additional N-acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-β-galactosidase treatment in order to release these tetra- and Sd a -active pentasaccharide. Differences were observed in the total amount of released tetra- and Sd a -active pentasaccharide of the used donors (42, 470, 478, 718 μg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd a determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the β-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N-acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N-acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd a determinants to structures containing no Sd a determinant (see below). One compound was used in order to specify the branch location of the additional N-acetyllactosamine unit, and it appeared that only the Gal-64 and Gal-84 residues were occupied by a repeating N-acetyllactosamine unit.

/pdf/the-abundance-of-additional-n-acetyllactosamine-units-in-n-7hsrlobnwu.pdf

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Uromodulin was isolated from urine of three pregnant women Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin Furthermore, no significant changes in the molar ratio between Man6GlcNAc2 and Man7GlcNAc2 were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences

/pdf/the-patterns-of-the-complex-and-oligomannose-type-glycans-of-3srce6eqkk.pdf

Johannes J.M. van Rooijen

Papers

Glycosylation sites and site-specific glycosylation in human Tamm-Horsfall glycoprotein

Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein.

Expression of N-linked sialyl Lex determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

The patterns of the complex- and oligomannose-type glycans of uromodulin (Tamm-Horsfall glycoprotein) in the course of pregnancy