Hybrid Oligosaccharides Research Articles

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAβ-1-3GlcNAc of the internal disaccharide units with GlcUAβ-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features

Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.

Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN308 and ASN409) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI–MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation.

N-glycan content modulates kainate receptor functional properties.

Ionotropic glutamate receptor (iGluR) subunits are N-glycosylated at 4-12 sites, and Golgi processing produces mature receptors that contain high-mannose, hybrid and complex oligosaccharides. N-glycosylation is crucial for receptor biogenesis, influences receptor trafficking and provides a binding site for carbohydrate binding proteins. Glycan moieties are large, polar and occasionally charged, and they are attached at sites along iGluRs that position them for involvement in the structural changes underlying gating. Altering glycan content on kainate receptors (KARs), a subfamily of iGluRs, changes functional properties of the receptor, such as desensitization, recovery from desensitization and deactivation. We report the first observation that the charged trisaccharide HNK-1 is conjugated to native KARs, and we find that it substantially alters recombinant KAR functional properties. Our results show that the molecular composition of N-glycans can influence KAR biophysical properties, revealing a potential mechanism for fine-tuning the function of these receptors. Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between four and 12 consensus sites for N-glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post-translational modification. N-glycosylation is required for proper folding of iGluRs in mammalian cells, although the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged and mediate many protein-protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. In the present study, we show that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide human natural killer-1. Glycosylation patterns probably differ between cell types, across development or with pathologies, and thus our findings reveal a potential mechanism for context-specific fine-tuning of KAR function through diversity in glycan structure.

Nonenzymatic Transglycosylation Reactions Induced by Roasting: New Insights from Models Mimicking Coffee Bean Regions with Distinct Polysaccharide Composition.

Three mixtures containing different molar proportions of (β1→4)-D-mannotriose and (α1→5)-L-arabinotriose, oligosaccharides structurally related to coffee polysaccharides (galactomannans and arabinogalactans), were roasted at 200 °C for different periods. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS(n)) analyses of labeled ((18)O) and unlabeled samples allowed identification of not only nonhybrid oligosaccharides but also hybrid oligosaccharides composed of both hexose and pentose units. The identification of hybrid oligosaccharides allowed us to infer the occurrence of nonenzymatic transglycosylation reactions involving both oligosaccharides in the starting mixtures. Also, it was observed that using different proportions of the oligosaccharides in the starting mixtures and extents of thermal treatment led to a variation in the composition of the compounds formed. These results have led to the conclusion that, depending on the distribution of the polysaccharides in the bean cell walls and the roasting conditions, different nonhybrid and hybrid structures can be formed during coffee roasting.

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System

The peptide-N (4)-(N-acetyl-β-D-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

Detection and quantitation of low abundance oligosaccharides in recombinant monoclonal antibodies.

Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics.

N‐glycan analysis of IgG by enzymatic digestion with Remove‐iT® Endo S and PNGase F (1004.1)

Objective: The objective of this study was to compare the release of N‐glycans from different sources of IgG using a novel endoglycosidase with a chitin‐binding domain tag (Remove‐iT Endo S) and PNGase F. Biotechnology companies are currently developing a growing number of monoclonal IgG antibodies as therapeutic agents. Critical for the structure and biological activity of the molecule is the N‐glycan moiety attached to the asparagine 297 residue in the constant domain of the antibody. There are many variables in cell culture systems that can greatly influence the heterogeneity of the glycans on IgG. For this reason it has become increasingly important to monitor the glycosylation profiles of these biotherapeutics in the production process.Methods: Remove‐iT Endo S (also known as Endoglycosidase S) was cloned from Streptococcus pyogenes and overexpressed in E. coli as a fusion to the chitin‐binding domain. IgG samples were enzymatically deglycosylated under native conditions using Remove‐iT Endo S and PNGase F Glycerol Free. The deglycosylated IgG samples were then analyzed by SDS‐PAGE gel shift analysis and ESI‐TOF MS to estimate the degree of deglycosylation. In addition, the glycans released from IgG following deglycosylation by both enzymes were analyzed using nano LC‐TOF MS.Results: Remove‐iT Endo S has a high specificity for removing the N‐glycan moiety of IgG after the first N‐acetylglucosamine (GlcNAc) residue, leaving only the GlcNAc with or without a core fucose residue. However, PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N‐linked glycoproteins.Conclusion: Remove‐iT Endo S is a more robust enzyme for the method described herein as it completely removes the sugar residues from IgG. Conversely, the PNGase F glycerol free digest does not result in a complete digestion under native conditions.

Protease-DeficientSaccharomycescerevisiaeStrains for the Synthesis of Human-Compatible Glycoproteins

Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man5GlcNAc2, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man8GlcNAc2 to Man5GlcNAc2 was observed after integration of Aspergillus saitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-β by approximately 10-fold.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Preparation of Hybrid Fucoidan-derived Oligosaccharides and Their Structural Analysis by Negative-ion Electrospray Tandem Mass Spectrometry

Acomplex sulfated fucoidan(SPF) was obtained by hot water extraction from Sargassum pallidum.A series of hybrid oligosaccharides were acquired by mild acid hydrolysis with low pressure gel permeation chromatography(LPGPC).Based on the monosaccharides composition,methylation and electrospray collision-induced dissociation mass spectrometry(ES-CID-MS/MS) analysis,21 kinds of oligosaccharides were sulfated hybrid fucoidan-derived oligosaccharides.The resulted oligosaccharides were mainly composed of α1→3 linked Fuc,β1→4 linked Xyl and β1→6 linked Gal.The sulfation occurred at C4 or C2 of fucose,C2 of xylose and C4 of galactose residue,respectively.Most of fucose residues were present in nonreducing terminal of oligosaccharides.The technique of ES-CID-MS/MS was suitable for the sequences analysis of other hybrid oligosaccharides of various fucoidans.More importantly,all these diverse sulfated oligosaccharides can be printed onto the glycol-chip,and studying their interactions with proteins.

Biochemical and Structural Characterization of the Complex Agarolytic Enzyme System from the Marine Bacterium Zobellia galactanivorans

Zobellia galactanivorans is an emerging model bacterium for the bioconversion of algal biomass. Notably, this marine Bacteroidetes possesses a complex agarolytic system comprising four β-agarases and five β-porphyranases, all belonging to the glycoside hydrolase family 16. Although β-agarases are specific for the neutral agarobiose moieties, the recently discovered β-porphyranases degrade the sulfated polymers found in various quantities in natural agars. Here, we report the biochemical and structural comparison of five β-porphyranases and β-agarases from Z. galactanivorans. The respective degradation patterns of two β-porphyranases and three β-agarases are analyzed by their action on defined hybrid oligosaccharides. In light of the high resolution crystal structures, the biochemical results allowed a detailed mapping of substrate specificities along the active site groove of the enzymes. Although PorA displays a strict requirement for C6-sulfate in the -2- and +1-binding subsites, PorB tolerates the presence of 3-6-anhydro-l-galactose in subsite -2. Both enzymes do not accept methylation of the galactose unit in the -1 subsite. The β-agarase AgaD requires at least four consecutive agarose units (DP8) and is highly intolerant to modifications, whereas for AgaB oligosaccharides containing C6-sulfate groups at the -4, +1, and +3 positions are still degraded. Together with a transcriptional analysis of the expression of these enzymes, the structural and biochemical results allow proposition of a model scheme for the agarolytic system of Z. galactanivorans.

Hyaluronan–chondroitin hybrid oligosaccharides as new life science research tools

Hyaluronan and chondroitin are glycosaminoglycans well-known as components of pharmaceutical agents and health foods. From these attractive molecules, using transglycosylation reaction of testicular hyaluronidase, we synthesized hybrid neo-oligosaccharides not found in nature. We also found a new site between the chondroitin disaccharide unit and hyaluronan disaccharide unit recognized by a hyaluronan lyase specific to hyaluronan using these hybrid oligosaccharides as substrates. We hope that these hybrid oligosaccharides will help to elucidate the involvement of hyaluronan, chondroitin, and chondroitin sulfates in the mechanisms of cell functions and diseases, based on the structures of these glycosaminoglycans.

L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s10719-012-9374-5) contains supplementary material, which is available to authorized users.

Structural and compositional characteristics of hybrid carrageenans from red algae Chondracanthus chamissoi

Two polysaccharides CCC and CCH were extracted from red algae Chondracanthus chamissoi by cold water and hot water, respectively. Characterization of the structure by chemical and spectroscopic methods showed that CCC was a hybrid carrageenan composed of κ-carrageenan (35%) and ι-carrageenan (43%) along with its precursor μ-carrageenan (22%) and CCH was an ideal κ-carrageenan. Oligosaccharides from CCH and CCC were prepared and their structural sequences determined by ES-CID-MS/MS gave further insight into the structural characteristics of hybrid carrageenans from C. chamissoi. Some hybrid oligosaccharides, e.g., κ–κ, κ–μ and κ–ι, were obtained with mild acid hydrolysis of CCC. Then, a regular even-numbered oligosaccharides generated with reductive acid hydrolysis of CCH confirmed it an ideal κ-carrageenan.

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

Lipooligosaccharide of Campylobacter jejuni: SIMILARITY WITH MULTIPLE TYPES OF MAMMALIAN GLYCANS BEYOND GANGLIOSIDES

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.

Structural analysis of the degradation products of porphyran digested by Zobellia galactanivorans β-porphyranase A

Porphyran from the red seaweed Porphyra umbilicalis was degraded with β-porphyranase A derived from the marine flavobacterium Zobellia galactanivorans. β-Porphyranase A produces a new series of sulfated oligosaccharides that we characterized by NMR, HPAEC and size-exclusion chromatography. In contrast to previously used β-agarases, which produce predominantly non-sulfated oligosaccharides of the neoagarobiose series and hybrid oligosaccharides, β-porphyranase A produced oligosaccharides of the α- l-Gal p-6-sulfate (1 → 3) ß- d-Gal p (L6S-G) series and the L6S-G disaccharide was the major final product. The newly identified β-porphyranase A was further used in combination with β-agarase B to revise porphyran polysaccharide structure.

Hybridity of carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula

Carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula were degraded by Pseudoalteromonas carrageenovora κ-carrageenase and Alteromonas fortis ι-carrageenase. The composition of the high molecular weight fraction (i.e., enzyme-resistant fraction) as well as the standard and hybrid oligosaccharide content highlighted differences between the species-specific carrageenans in terms of composition and distribution of the carrabiose moieties. Inspection of the distribution of μ- and ν-carrabiose, the biosynthetic precursors of κ- and ι-carrabiose, revealed their localization in κ-carrabiose- or ι-carrabiose-rich segments, respectively.