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Synthesis and processing of thyroglobulin carbohydrate units
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STRUCTURE OF HO~ONE-CONTAINING PEPTIDES IN PORCINE AND HUMAN ~R~L~LINS C. Marriq, P.J. Lejeune, M, Rolland and S. Lissitsky, U 38 INSERM, Faculte de MBdecine 27 Bd J. Moulin, 13385 Marseille Cddex 05, France. In vitro and in vivo iodination studies on thyroglobulin (Tgb) isolated from different species showed that of the about 120 tyrosine residues contained in the protein only 8 to 10 are involved in hormone formation (T4 and T3). Determination of the primary structure of Tgb has recently been the object of several investigations using sequencing of the cloned cDNA to Tgb mRNA. However additional knowledge of the aminoacid sequence around the hormone residues in the mature protein is needed to locate the tyrosine residues involved in thyroid hormone biosynthesis, Such sequences have been studied in porcine Tgb of high iodine content (about 1% I, w/w) and poorly iodinated human Tgb (0,2% I, w/w). In porcine Tgb after CNBr cleavage and purification of the CNBr-peptides, 4 discrete hormono-peptidesWre sequenced. Two of them : His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-TyrPhe-Ser-Ser-Arg and Glu-T4-Gln-Val-Aspcorrespond to sites of high affinity to form thyroid hormones whereas the other two : Asp-T4-Phe-Ile-Leu-XPro-Val and Leu-Ala-Ser-Lys-Ser-T4fT3are effective only for higher iodination levels. In human Tgb a naturally occuring 18 KDa peptide released after reduction of the molecule was purified ; it corresponds to the Nterminal part of the mature Tgb where is located an hormonogenic site in the sequence : Asn-Ile-Phe-Glu-T4-Gin-Val-Asp.This site which contains about 50 % of the total T4 of the molecule has a structure identical to one of the high affinity site found in porcine Tgb.
Cl0 SYNTHESIS AND PROCESSING OF THYROGLOBULIN CARBOHYDRATE UNITS. Mary Jane m* and Robert G. Spiro, Harvard Medical School, Joslin Research Laboratory, Boston, Massachusetts, U.S.A. In the course of the synthesis of thyroglobulin,multiple carbohydrate units (20 per molecule in calf) consisting of Glc3MangGlcNAc2 are added cotranslationally to Asn residues of this protein from a dolichyl (Dal)P-P-oligosaccharidelocated in the rough endoplasmic reticulum (RER). Approximately one-third of these remain as the polymannose type (Unit A) while the remainder are converted to complex units (Unit B). Processing begins in the RER with removal of the Glc and the first Man residue; after translocation of the protein to the Golgi additional Man is removed and 2-4 oligosaccharide chains are formed consisting of G~cNAc, Gal, and NeuAc. Species variation in thyroglobulin carbohydrate is marked, with a-Gal residues occurring in Unit B of calf, sheep and pig but not man; a third type of unit linked to Thr(Ser) through GalNAc is present in man and guinea pig; while in the human protein a uranic acid-containing glycosaminoglycan-like polymer also occurs. For transfer to Asn residues, the Glc sequence of the oligosaccharide-lipiddonor is essential and addition of this sugar is highly sensitive to the energy level of the cell, with the lipid-linked ManB_gGlcNAcg intermediates accumulating during ATP depletion. Availabillty of Dol-P is also a determinant of oligosaccharide-lipidsynthesis, with protein glycosylation exerting a positive feedback by releasing free Dol-P. Tightly coupled enzymes have been found in the thyroid RER which are responsible for translocation of sugars (Glc,Man,GlcNAc)from their nucleotides, synthesis of the Dol-P-P-oligosaccharidedonor, and transfer of the completed oligosaccharide to newly synthesized protein.
c9
STRUCTURE OF HO~ONE-CONTAINING PEPTIDES IN PORCINE AND HUMAN ~R~L~LINS C. Marriq, P.J. Lejeune, M, Rolland and S. Lissitsky, U 38 INSERM, Faculte de MBdecine 27 Bd J. Moulin, 13385 Marseille Cddex 05, France. In vitro and in vivo iodination studies on thyroglobulin (Tgb) isolated from different species showed that of the about 120 tyrosine residues contained in the protein only 8 to 10 are involved in hormone formation (T4 and T3). Determination of the primary structure of Tgb has recently been the object of several investigations using sequencing of the cloned cDNA to Tgb mRNA. However additional knowledge of the aminoacid sequence around the hormone residues in the mature protein is needed to locate the tyrosine residues involved in thyroid hormone biosynthesis, Such sequences have been studied in porcine Tgb of high iodine content (about 1% I, w/w) and poorly iodinated human Tgb (0,2% I, w/w). In porcine Tgb after CNBr cleavage and purification of the CNBr-peptides, 4 discrete hormono-peptidesWre sequenced. Two of them : His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-TyrPhe-Ser-Ser-Arg and Glu-T4-Gln-Val-Aspcorrespond to sites of high affinity to form thyroid hormones whereas the other two : Asp-T4-Phe-Ile-Leu-XPro-Val and Leu-Ala-Ser-Lys-Ser-T4fT3are effective only for higher iodination levels. In human Tgb a naturally occuring 18 KDa peptide released after reduction of the molecule was purified ; it corresponds to the Nterminal part of the mature Tgb where is located an hormonogenic site in the sequence : Asn-Ile-Phe-Glu-T4-Gin-Val-Asp.This site which contains about 50 % of the total T4 of the molecule has a structure identical to one of the high affinity site found in porcine Tgb.
Cl0 SYNTHESIS AND PROCESSING OF THYROGLOBULIN CARBOHYDRATE UNITS. Mary Jane m* and Robert G. Spiro, Harvard Medical School, Joslin Research Laboratory, Boston, Massachusetts, U.S.A. In the course of the synthesis of thyroglobulin,multiple carbohydrate units (20 per molecule in calf) consisting of Glc3MangGlcNAc2 are added cotranslationally to Asn residues of this protein from a dolichyl (Dal)P-P-oligosaccharidelocated in the rough endoplasmic reticulum (RER). Approximately one-third of these remain as the polymannose type (Unit A) while the remainder are converted to complex units (Unit B). Processing begins in the RER with removal of the Glc and the first Man residue; after translocation of the protein to the Golgi additional Man is removed and 2-4 oligosaccharide chains are formed consisting of G~cNAc, Gal, and NeuAc. Species variation in thyroglobulin carbohydrate is marked, with a-Gal residues occurring in Unit B of calf, sheep and pig but not man; a third type of unit linked to Thr(Ser) through GalNAc is present in man and guinea pig; while in the human protein a uranic acid-containing glycosaminoglycan-like polymer also occurs. For transfer to Asn residues, the Glc sequence of the oligosaccharide-lipiddonor is essential and addition of this sugar is highly sensitive to the energy level of the cell, with the lipid-linked ManB_gGlcNAcg intermediates accumulating during ATP depletion. Availabillty of Dol-P is also a determinant of oligosaccharide-lipidsynthesis, with protein glycosylation exerting a positive feedback by releasing free Dol-P. Tightly coupled enzymes have been found in the thyroid RER which are responsible for translocation of sugars (Glc,Man,GlcNAc)from their nucleotides, synthesis of the Dol-P-P-oligosaccharidedonor, and transfer of the completed oligosaccharide to newly synthesized protein.