Gavin M. Brown

Human aggrecan keratan sulfate undergoes structural changes during adolescent development

Alkaline borohydride-reduced keratan sulfate chains were isolated from human articular cartilage aggrecan from individuals of various ages (0–85 years old). The chains were structurally characterized using 1H NMR spectroscopy, gel permeation chromatography, and oligosaccharide profiling (after digestion with the enzymes keratanase and keratanase II). The results show that from birth to early adolescence (0–9 years) the levels of α(1–3)-fucosylation, α(2–3)-sialylation, and galactose sulfation increase. Also, the weight-average molecular weight of the chains increases. During maturation (9–18 years) the levels of fucosylation and galactose sulfation continue to increase and α(2–6)-sialylation of the chains occurs. In adult life (18–85 years) there is little change in the weight-average molecular weight of the chains, and the levels of fucosylation, sialylation, and sulfation remain fairly constant.

Proteomic and Postproteomic Characterization of Keratan Sulfate-Glycanated Isoforms of Thyroglobulin and Transferrin Uniquely Elaborated by Papillary Thyroid Carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.

A full assignment of proton resonances for an alpha(1-3)-linked fucose residue in keratan sulphate from bovine articular cartilage.

Full proton NMR assignments have been achieved for the α(1-3)-linked fucose residues contained in alkaline borohydride reduced keratan sulphate chains derived from bovine articular cartilage. This involved 500 MHz spectroscopy at 60°C and included COSY and RELAYED-COSY determinations.

13C-NMR spectroscopy of keratan sulphates. Assignments for four poly(N-acetyllactosamine)-repeat-sequence tetrasaccharides derived from bovine articular cartilage keratan sulphate by keratanase II digestion.

Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and the complete 13C chemical shift data are reported for the four tetrasaccharides which derived from the repeat region. They have the structures: Gal beta(1-4)GlcNAc(6S)beta(1-3)Gal beta(1-4)GlcNAc(6S)-ol, Gal(6S)beta(1-4)GlcNAc(6S)beta(1-3)Gal(1-4)GlcNAc(6S)-ol, Gal beta(1-4)GlcNAc(6S)beta(1-3)Gal(6S)beta(1-4)GlcNAc(6S)-ol, and Gal(6S)beta(1-4)GlcNAc(6S)(1-3)Gal(6S)beta(1-4)GlcNAc(6S)-ol, where GlcNAc(6S)-ol represents N-acetyl-glucosaminitol-6-O-sulphate. The value of these data for microstructural analysis of undegraded keratan sulphate samples using 13C-NMR spectroscopy is discussed with respect to galactose sulphation levels and the block sulphation structure.

Keratanase digestion of keratan sulphates: characterization of large oligosaccharides from the N-acetyllactosamine repeat sequence and from the non-reducing terminal chain caps.

Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography. The main beta-linked pentasulfated hexasaccharide repeat segment, [R6], GlcNAc(6S)1-1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal-ol and the asialo beta-linked capping pentasulfated heptasaccharide, [C7], Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) 1-3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods. Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-5(1-3)Gal-0l [R8,R10,R12] Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]3-5(1-3)Gal-ol [C9,C11,C13] NeuAc alpha 2-3Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-4(1-3)Gal-ol [C8,C10,C12] obtained from keratan sulfate by keratanase cleavage. The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths. The others, viz. those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Oligosaccharide Profiling of Keratan Sulphate

Keratan sulphate (KS) was first isolated from bovine cornea (1), and was subsequently also extracted from the nucleus pulposus of human intervertebral disc (2). Since then, KS chains have been extracted from a number of tissues, and the following KS-containing proteoglycans have been identified: aggrecan (3) from cartilage, lumican (4) from cornea, two other corneal KS-Proteoglycans (PGs) with core protein sizes of 37 kDa and 25 kDa (5), fibromodulin (6) from various tissues, abakan (7), claustrin (8), phosphacan (9) and neurocan (10) from brain, SV2 (11) from synaptic vesicles etc. In addition, other molecules closely related to KS have been characterized, such as those from the zona pellucida (12).

An ultrastructural investigation of dermatopontin-knockout mouse corneas

Introduction  The corneal stroma is composed of a network of heterotypic collagen fibrils, proteoglycans and matrix proteins. Transparency of the tissue principally requires the uniformity of fibril diameters and interfibrillar distances and the presence of a quasi‐hexagonal lattice arrangement of parallel fibrils. Keratan sulfate proteoglycans (KSPGs) have a crucial role and the KS chains are clearly required for the maintenance of transparency. Undersulfation of corneal KS results in tissue opacity and the lumican (a KSPG) knockout mouse shows corneal opacity and the disruption of collagen fibril diameters and interfibrillar distances ( Chakravarti et al. 1998 ). Biochemical analysis has shown that dermatopontin is an abundant component of the extracellular matrix and that it interacts with KSPGs via the KS chains. This study aims to determine whether dermatopontin has a direct role in corneal matrix organization by investigating the corneal ultrastructure of dermatopontin‐null (dpt–/–) mouse corneas.

Fabrication of Carbohydrate Surfaces by Using Non-derivatised Oligosaccharides

Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index, and mass of adsorbates 100-nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates are suitable for studying their interactions with proteins and where non-specific binding is reduced to less than 5% of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional cross-linker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulphate, chondroitin sulphate, and heparin that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG, and the surfaces were then challenged with a variety of proteins.