Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles.

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Oligosaccharides derived by endo-beta-galactosidase digestion of bovine corneal keratan sulphate--characterisation of tetrasaccharides with incomplete sulphation and containing unsulphated N-acetylglucosamine residues.

Bovine corneal keratan sulphate has been fragmented using the enzyme endo-beta)-galactosidase and 1H-NMR chemical shift data are reported for five newly isolated tetrasaccharides which derived from the repeat region. They have the structures: GlcNAc(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal-ol, GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal6S(beta1-4)GlcNAc(beta1-3)Gal-ol, and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol. Structures for two trisaccharides formed by a peeling reaction are also given. These are GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc-ol and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S-ol where GlcNAc6S-ol represents N-acetylglucosaminitol 6-O-sulphate. Characterisation of such structures and their spectral assignments will be of considerable value for the studies of both selectin ligands and undersulphated keratan sulphates such as those occurring on cell surfaces and in brain tissue.

Human corneal keratan sulfates.

The keratan sulfate-containing proteoglycans were isolated from fourteen pooled human corneas (thirteen from 61- to 86-year-olds, plus one from a 12-year-old). These proteoglycans were subjected to digestion with the enzyme keratanase II, and the released oligosaccharides, which included nonreducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and identified on two separate ion-exchange columns. Both of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol. Chem. 271, 23535-23546) and all of which had been fully characterized by NMR spectroscopic analysis. The capping structures identified in human corneal keratan sulfates occurred in the relative proportions: NeuAcalpha(2-6)- >NeuAcalpha(2-3)- >GalNAc(S)beta(1-3)-. The other groups of capping structures which had been identified in bovine corneal keratan sulfate, i.e. NeuGcalpha(2-3)-, NeuGcalpha(2-6)-, GlcNAc(S)beta(1-3)- were absent, although the possibility of the presence of some Galalpha(1-3)- structures could not be excluded. In addition, the human sample showed significantly higher levels of alpha(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age dependence as the bovine corneas were from young animals, whereas the human corneas were predominantly from an older group. The charge densities and keratan sulfate chain sizes of the human and bovine keratan sulfate-containing proteoglycans were seen to be similar.

Multiple non-reducing chain termini isolated from bovine corneal keratan sulfates.

Keratan sulfate-containing proteoglycans were isolated from bovine cornea (15-month-old to 3-year-old animals) and digested with the enzyme, keratanase II. The released oligosaccharides, which included non-reducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and fractionated on a Spherisorb column. These oligosaccharides were examined by 600-MHz 1H NMR spectroscopy using one- and two-dimensional methods and, in addition to some oligosaccharide alditols previously recovered from skeletal keratan sulfate, the following new capping structures were identified: NeuAcalpha2-6Galbeta1-4GlcNAc(S)-ol, NeuAcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-6Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-o l, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc( S)-ol, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)- ol, GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol, and GalNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol. These structures represent seven families of capping residues, whose relative molar proportions are given in parentheses: NeuAcalpha(2-3)- (12%), NeuAcalpha(2-6)- (41%), NeuGcalpha(2-3)- and NeuGcalpha(2-6)- families (12%), Galalpha(1-3)- (26%), GalNAc(S)beta(1-3)- (5%), and GlcNAc(S)beta(1-3)- (4%). It is not clear, at present, where each of these structures occurs on the bi-antennary N-linked corneal keratan sulfate chains, which themselves occur within three keratan sulfate proteoglycan species. However, examination of the relative proportions of the capping to the repeat structures and knowledge of the average molecular size suggests that the sum of these non-reducing termini represents the caps of two antennae.

N.m.r. spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates. Influence of fucose residues on keratanase cleavage.

Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp. and the products were reduced with alkaline borohydride. The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated. Structural analysis using 1H n.m.r. spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol. Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated. Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage. This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses. It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose.

A sub-population of keratan sulphates derived from bovine articular cartilage is capped with alpha(2-6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy.

Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids.

Fucose content of keratan sulphates from bovine articular cartilage.

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals). Nine keratan sulphate fractions of increasing molecular weight were prepared by gel-permeation chromatography on a calibrated column of TSK 30 XL. The samples were analysed for fucose and galactose contents (% by wt. of keratan sulphate) and fucose/galactose ratio. The fucose content increased with molecular size, but the galactose content remained constant. It was concluded that the alpha(1----3)-linked fucose [Thornton, Morris, Cockin, Huckerby, Nieduszynski, Carlstedt, Hardingham & Ratcliffe (1989) Biochem. J. 260, 277-282] was located within the poly-N-acetyl-lactosamine repeat sequence of articular-cartilage keratan sulphate.

There are two major types of skeletal keratan sulphates.

High-field 1H-n.m.r.-spectroscopic studies supported by chemical carbohydrate analyses show that skeletal keratan sulphates (KS-II) of bovine origin may be sub-classified into two groups. Keratan sulphate chains from articular and intervertebral-disc cartilage (KS-II-A) contain two structural features, namely alpha(1----3)-fucose and alpha(2----6)-linked N-acetyl-neuraminic acid residues, that are absent from keratan sulphates from tracheal or nasal-septum cartilage (KS-II-B).