Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.
Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.
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DOI：
10.1186/1471-2091-12-6
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发表时间：
2011-02-03
期刊：
影响因子：
--
通讯作者：
Simmer JP
中科院分区：
文献类型：
--
作者：
Yamakoshi Y;Nagano T;Hu JC;Yamakoshi F;Simmer JP
牙本质涎磷蛋白（Dspp）是一种多结构域的分泌性蛋白质，对牙本质的形成至关重要。DSPP的突变导致遗传性牙本质缺陷，分为牙本质发育不良II型和牙本质发生异常II型和III型。牙本质涎蛋白（Dsp）是牙本质涎磷蛋白（Dspp）的N-末端结构域，是一种高度糖基化的蛋白多糖，但其糖基的数量、性质和连接位点尚不清楚。为了确定其碳水化合物的附着位点，我们分离出Dsp从发展中的猪磨牙和内切蛋白酶Glu-C或链霉蛋白酶消化，分馏的消化产物，确定馏分含有糖基化肽使用苯酚硫酸测定，其特征在于糖肽的N-末端测序，氨基酸分析，或LC/MSMS。为了确定每个N-糖基化的唾液酸附着的平均数，我们用糖肽酶A消化Dsp，用2-氨基苯甲酸标记释放的N-糖基化，并通过与已知浓度的标记标准品比较来定量释放的糖基化的摩尔数。通过唾液酸酶消化释放唾液酸，并通过测量β-NADH还原的唾液酸定量，所述β-NADH还原是通过醛缩酶从唾液酸化学计量产生的。为了确定其形式，用1，2-二氨基-4，5-亚甲基氧基苯（DMB）标记唾液酸酶消化释放的唾液酸，并通过RP-HPLC与DMB标记的唾液酸参比组进行比较。为了确定Dsp糖胺聚糖（GAG）附件的组成，我们用软骨素酶ABC消化Dsp，并将释放的二糖的色谱图与商业标准品进行比较。在Asn 37、Asn 77、Asn 136、Asn 155、Asn 161和Asn 176处鉴定了N-糖基化。Dsp平均每个N-糖基化一个唾液酸，其总是以N-乙酰神经氨酸的形式存在。O-糖基化暂时位于Thr 200、Thr 216和Thr 316。在Ser 238和Ser 250处发现猪Dsp GAG附着，其由比例分别为7：3的6-硫酸软骨素和4-硫酸软骨素组成。猪Dsp翻译后修饰的分布表明，猪Dsp具有至少6个N-糖基化的N-末端结构域和至少2个O-糖基化的2个GAG连接的C-末端结构域。
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. 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 Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr316. Porcine Dsp GAG attachments were found at Ser238 and Ser250 and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively. 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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影响因子：
4.2
作者：
LINDE, A;LUSSI, A;CRENSHAW, MA
通讯作者：
CRENSHAW, MA
影响因子：
7.6
作者：
McKnight DA;Simmer JP;Hart PS;Hart TC;Fisher LW
通讯作者：
Fisher LW
影响因子：
1.9
作者：
Kida, Miyuki;Tsutsumi, Tomonori;Ariga, Tadashi
通讯作者：
Ariga, Tadashi
影响因子：
7.6
作者：
Lee, S. -K.;Lee, K. -E.;Kim, J. -W.
通讯作者：
Kim, J. -W.
影响因子：
5.3
作者：
Kim, JW;Hu, JCC;Simmer, JP
通讯作者：
Simmer, JP