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Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro.

Chen Y, Dong W, Tan L, Held MA, Kieliszewski MJ - Biochem Insights (2015)

Bottom Line: Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking.Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate.Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USA.

ABSTRACT
Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

No MeSH data available.


Structure elucidation of RSH Hyp-Ara4. (A) HSQC spectrum: cross-peaks identified the chemical shifts of each carbon atom and its corresponding hydrogen atom(s) in each arabinose ring system. A4 is the fourth Ara at the non-reducing end of the Hyp-Ara4 chain, while A1 occupies the reducing end and is attached to Hyp (first Ara in the chain). A2 and A3 are the second and third Ara of the chain. The A1 C1/H1 label indicates the cross-peak arising from the chemical shifts of the anomeric carbon (C1) and its corresponding hydrogen (H1) on the A1 residue. The cross-peaks for the other carbon atoms and their corresponding hydrogens of A1 and the cross-peaks for A2 to A4 are similarly labeled. Two cross-peaks are observed for the fifth carbon atoms on each ring system due to their possession of two corresponding hydrogen atoms. (B) HMBC spectrum: the cross-peaks arising from A4H1(5.2 ppm) + A3C3(84.6 ppm) and A4C1(112.1 ppm) + A3H3(4.2 ppm), highlighted by red circles, established the α-Araf-(1→3)-β-Araf linkage between A4 and A3; cross-peaks arising from A3H1(5.1 ppm) + A2C2(84.5 ppm) and A3C1(103.8 ppm) + A2H2(4.3 ppm), highlighted by blue circles, established the β-Araf-(1→2)-β-Araf linkage between A3 and A2; cross-peaks arising from A2H1(5.2 ppm) + A1C2(84.5 ppm) and A2C1(102.7 ppm) + A1H2(4.3 ppm), highlighted by orange circles, established the β-Araf-(1→2)-β-Araf linkage between A2 and A1, and cross-peak arising from A1H1(5.3 ppm) + HypC4 (81.1 ppm), highlighted by the green circle, established the β-Araf-(1→4)-Hyp linkage between A1 and Hyp. The chemical shifts of the cross-peaks are summarized in Table 5.
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f4-bci-suppl.2-2015-001: Structure elucidation of RSH Hyp-Ara4. (A) HSQC spectrum: cross-peaks identified the chemical shifts of each carbon atom and its corresponding hydrogen atom(s) in each arabinose ring system. A4 is the fourth Ara at the non-reducing end of the Hyp-Ara4 chain, while A1 occupies the reducing end and is attached to Hyp (first Ara in the chain). A2 and A3 are the second and third Ara of the chain. The A1 C1/H1 label indicates the cross-peak arising from the chemical shifts of the anomeric carbon (C1) and its corresponding hydrogen (H1) on the A1 residue. The cross-peaks for the other carbon atoms and their corresponding hydrogens of A1 and the cross-peaks for A2 to A4 are similarly labeled. Two cross-peaks are observed for the fifth carbon atoms on each ring system due to their possession of two corresponding hydrogen atoms. (B) HMBC spectrum: the cross-peaks arising from A4H1(5.2 ppm) + A3C3(84.6 ppm) and A4C1(112.1 ppm) + A3H3(4.2 ppm), highlighted by red circles, established the α-Araf-(1→3)-β-Araf linkage between A4 and A3; cross-peaks arising from A3H1(5.1 ppm) + A2C2(84.5 ppm) and A3C1(103.8 ppm) + A2H2(4.3 ppm), highlighted by blue circles, established the β-Araf-(1→2)-β-Araf linkage between A3 and A2; cross-peaks arising from A2H1(5.2 ppm) + A1C2(84.5 ppm) and A2C1(102.7 ppm) + A1H2(4.3 ppm), highlighted by orange circles, established the β-Araf-(1→2)-β-Araf linkage between A2 and A1, and cross-peak arising from A1H1(5.3 ppm) + HypC4 (81.1 ppm), highlighted by the green circle, established the β-Araf-(1→4)-Hyp linkage between A1 and Hyp. The chemical shifts of the cross-peaks are summarized in Table 5.

Mentions: TFA elution of RSH Hyp-oligoarabinosides yielded a profile similar to that of HCl elution (Fig. 1A, first panel). Peaks containing Hyp-Ara4 and Hyp-Ara3 were collected (cis and trans peaks pooled for NMR analysis), and through NMR spectra (COSY, TCOSY, HSQC, and HMBC) the glycosyl residues, ring systems, and ordered linkages of the components of RSH Hyp-Ara4 (Fig. 4) and Hyp-Ara3 (Fig. 5) were identified. The results corroborate the chemical shift obtained earlier by Akiyama et al50,51 for Hyparabinosides isolated from tobacco suspension culture cell walls (Table 5) and identified the structures of RSH Hyp-Ara4 and Hyp-Ara3 as (Fig. 6).


Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro.

Chen Y, Dong W, Tan L, Held MA, Kieliszewski MJ - Biochem Insights (2015)

Structure elucidation of RSH Hyp-Ara4. (A) HSQC spectrum: cross-peaks identified the chemical shifts of each carbon atom and its corresponding hydrogen atom(s) in each arabinose ring system. A4 is the fourth Ara at the non-reducing end of the Hyp-Ara4 chain, while A1 occupies the reducing end and is attached to Hyp (first Ara in the chain). A2 and A3 are the second and third Ara of the chain. The A1 C1/H1 label indicates the cross-peak arising from the chemical shifts of the anomeric carbon (C1) and its corresponding hydrogen (H1) on the A1 residue. The cross-peaks for the other carbon atoms and their corresponding hydrogens of A1 and the cross-peaks for A2 to A4 are similarly labeled. Two cross-peaks are observed for the fifth carbon atoms on each ring system due to their possession of two corresponding hydrogen atoms. (B) HMBC spectrum: the cross-peaks arising from A4H1(5.2 ppm) + A3C3(84.6 ppm) and A4C1(112.1 ppm) + A3H3(4.2 ppm), highlighted by red circles, established the α-Araf-(1→3)-β-Araf linkage between A4 and A3; cross-peaks arising from A3H1(5.1 ppm) + A2C2(84.5 ppm) and A3C1(103.8 ppm) + A2H2(4.3 ppm), highlighted by blue circles, established the β-Araf-(1→2)-β-Araf linkage between A3 and A2; cross-peaks arising from A2H1(5.2 ppm) + A1C2(84.5 ppm) and A2C1(102.7 ppm) + A1H2(4.3 ppm), highlighted by orange circles, established the β-Araf-(1→2)-β-Araf linkage between A2 and A1, and cross-peak arising from A1H1(5.3 ppm) + HypC4 (81.1 ppm), highlighted by the green circle, established the β-Araf-(1→4)-Hyp linkage between A1 and Hyp. The chemical shifts of the cross-peaks are summarized in Table 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4-bci-suppl.2-2015-001: Structure elucidation of RSH Hyp-Ara4. (A) HSQC spectrum: cross-peaks identified the chemical shifts of each carbon atom and its corresponding hydrogen atom(s) in each arabinose ring system. A4 is the fourth Ara at the non-reducing end of the Hyp-Ara4 chain, while A1 occupies the reducing end and is attached to Hyp (first Ara in the chain). A2 and A3 are the second and third Ara of the chain. The A1 C1/H1 label indicates the cross-peak arising from the chemical shifts of the anomeric carbon (C1) and its corresponding hydrogen (H1) on the A1 residue. The cross-peaks for the other carbon atoms and their corresponding hydrogens of A1 and the cross-peaks for A2 to A4 are similarly labeled. Two cross-peaks are observed for the fifth carbon atoms on each ring system due to their possession of two corresponding hydrogen atoms. (B) HMBC spectrum: the cross-peaks arising from A4H1(5.2 ppm) + A3C3(84.6 ppm) and A4C1(112.1 ppm) + A3H3(4.2 ppm), highlighted by red circles, established the α-Araf-(1→3)-β-Araf linkage between A4 and A3; cross-peaks arising from A3H1(5.1 ppm) + A2C2(84.5 ppm) and A3C1(103.8 ppm) + A2H2(4.3 ppm), highlighted by blue circles, established the β-Araf-(1→2)-β-Araf linkage between A3 and A2; cross-peaks arising from A2H1(5.2 ppm) + A1C2(84.5 ppm) and A2C1(102.7 ppm) + A1H2(4.3 ppm), highlighted by orange circles, established the β-Araf-(1→2)-β-Araf linkage between A2 and A1, and cross-peak arising from A1H1(5.3 ppm) + HypC4 (81.1 ppm), highlighted by the green circle, established the β-Araf-(1→4)-Hyp linkage between A1 and Hyp. The chemical shifts of the cross-peaks are summarized in Table 5.
Mentions: TFA elution of RSH Hyp-oligoarabinosides yielded a profile similar to that of HCl elution (Fig. 1A, first panel). Peaks containing Hyp-Ara4 and Hyp-Ara3 were collected (cis and trans peaks pooled for NMR analysis), and through NMR spectra (COSY, TCOSY, HSQC, and HMBC) the glycosyl residues, ring systems, and ordered linkages of the components of RSH Hyp-Ara4 (Fig. 4) and Hyp-Ara3 (Fig. 5) were identified. The results corroborate the chemical shift obtained earlier by Akiyama et al50,51 for Hyparabinosides isolated from tobacco suspension culture cell walls (Table 5) and identified the structures of RSH Hyp-Ara4 and Hyp-Ara3 as (Fig. 6).

Bottom Line: Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking.Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate.Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USA.

ABSTRACT
Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

No MeSH data available.