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Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

BACKGROUND: Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp),...

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Autores principales: Yamakoshi, Yasuo, Nagano, Takatoshi, Hu, Jan CC, Yamakoshi, Fumiko, Simmer, James P
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039539/
https://www.ncbi.nlm.nih.gov/pubmed/21291557
http://dx.doi.org/10.1186/1471-2091-12-6
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author Yamakoshi, Yasuo
Nagano, Takatoshi
Hu, Jan CC
Yamakoshi, Fumiko
Simmer, James P
author_facet Yamakoshi, Yasuo
Nagano, Takatoshi
Hu, Jan CC
Yamakoshi, Fumiko
Simmer, James P
author_sort Yamakoshi, Yasuo
collection PubMed
description BACKGROUND: Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. RESULTS: To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn(37), Asn(77), Asn(136), Asn(155), Asn(161), and Asn(176). Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr(200), Thr(216 )and Thr(316). Porcine Dsp GAG attachments were found at Ser(238 )and Ser(250 )and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively. CONCLUSIONS: The distribution of porcine Dsp posttranslational modifications indicate that porcine Dsp has an N-terminal domain with at least six N-glycosylations and a C-terminal domain with two GAG attachments and at least two O-glycosylations.
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spelling pubmed-30395392011-02-16 Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments Yamakoshi, Yasuo Nagano, Takatoshi Hu, Jan CC Yamakoshi, Fumiko Simmer, James P BMC Biochem Research Article BACKGROUND: Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. RESULTS: To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn(37), Asn(77), Asn(136), Asn(155), Asn(161), and Asn(176). Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr(200), Thr(216 )and Thr(316). Porcine Dsp GAG attachments were found at Ser(238 )and Ser(250 )and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively. CONCLUSIONS: The distribution of porcine Dsp posttranslational modifications indicate that porcine Dsp has an N-terminal domain with at least six N-glycosylations and a C-terminal domain with two GAG attachments and at least two O-glycosylations. BioMed Central 2011-02-03 /pmc/articles/PMC3039539/ /pubmed/21291557 http://dx.doi.org/10.1186/1471-2091-12-6 Text en Copyright ©2011 Yamakoshi et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Yamakoshi, Yasuo
Nagano, Takatoshi
Hu, Jan CC
Yamakoshi, Fumiko
Simmer, James P
Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title_full Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title_fullStr Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title_full_unstemmed Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title_short Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
title_sort porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039539/
https://www.ncbi.nlm.nih.gov/pubmed/21291557
http://dx.doi.org/10.1186/1471-2091-12-6
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