Selectivity in tyrosyl iodination sites in human thyroglobulin.

Previous studies indicate that when low iodine thyroglobulin (Tg) is iodinated enzymatically with thyroid peroxidase (TPO), the tyrosyl residues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the enzyme (TPO) in the selection of the early tyrosyl sites that undergo iodination. For this purpose, low iodine human Tg (2.0 atoms I per 660,000 dimer) was iodinated chemically with (125)I-(3) and enzymatically with TPO + 125I- to a matched low level of iodination (approximately 8 added I atoms per molecule). After reduction and alkylation, the two Tg preparations were digested with trypsin, and the tryptic digests were separated by reverse-phase HPLC into 10 125I-containing pools. Each pool was further fractionated by HPLC to provide purified 125I-peptides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the location of the iodinated tyrosyl residues. Surprisingly, almost identical results were obtained with chemically and enzymatically iodinated Tg. Not only were the 125I-peptide maps very similar, but all of the recovered 125I in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Tyr 2553 are well-established sites of thyroxine formation, while Tyr 2520 has previously been proposed by us to be a donor site. Our observation that the same hormonogenic tyrosyl sites are iodinated by chemical as well as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyrosyl iodination sites in Tg. Consistent with this conclusion was our finding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centrifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (DIT) content and a correspondingly lower monoiodotyrosine content in enzymatically iodinated Tg, compared to matched chemically iodinated Tg. This was not observed with two other proteins, bovine serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, however, observed when lactoperoxidase was substituted for TPO. Preferential formation of DIT, therefore, appears to involve interaction between Tg and the peroxidase.

Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin.

The amino acid sequence established for human thyroglobulin (hTG) from its cDNA sequence contains 20 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate. In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined. Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC. The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin. Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation. Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein. Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine. Five sites (at positions 1200, 1329, 1993, 2275, and 2562) contain high mannose type units and two sites (at positions 179 and 1345) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures. In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence. A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites. The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin (bTG) (27) and as has been recently reported for bTG, no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG. Only four of the 20 putative sites the sequence of hTG, at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation.

Characterization of hormonogenic sites in an N-terminal, cyanogen bromide fragment of human thyroglobulin.

We have confirmed the observation of Marriq et al. (FEBS Lett. 207, 302-306, 1986) that substantial thyroxine formation occurs on enzymatic iodination of a 171-residue, N-terminal cyanogen bromide (CNBr) fragment of human goiter thyroglobulin. Marriq et al. concluded from their studies that Tyr130 is the donor for the preferential hormonogenic acceptor site Tyr5. However, in the present study we observed that thyroxine formation in the CNBr fragment occurred not at Tyr5, but rather at Tyr130. Moreover, we did not confirm the report of Marriq et al. that formation of thyroxine in the iodinated CNBr fragment involves cleavage of the Val129-Tyr130 bond. Our finding of thyroid hormone at Tyr130 was unexpected, as Tyr5 is known to be the major site of hormone formation in thyroglobulin isolated from the thyroids of humans and other mammals. In the CNBr fragment, however, Tyr5 appeared to act as a donor rather than an acceptor. Thus, while we have confirmed the finding that the N-terminal CNBr fragment of human goiter thyroglobulin is by itself capable of efficient thyroxine formation when enzymatically iodinated in vitro, we observed that the role of specific tyrosines in the hormone-forming process was different from that in intact thyroglobulin. We question, therefore, whether the CNBr fragment is a valid model for defining the role and location of hormonogenic sites in intact thyroglobulin.

Effect of peptide acetylation on its interaction with antipeptide antibody.

The effects of trace acetylation on the contact sites in peptide-antibody binding have been investigated by using a label selection method. A 13-residue synthetic peptide having three lysines (LKLVEQQNPKVKL) corresponding to sequence position 155-168 of beta 1,4-galactosyltransferase was used in this study. The four amino groups located throughout the peptide sequence, labeled appropriately, served as probing marker groups. The amino terminus region of the peptide was highly reactive to trace acetylation. Over 80% of the label appeared in the amino terminus region, most likely in the alpha-amino group due to its lower pK value. Interestingly, acetylation of Lys10 and Lys12 (carboxy terminal region) showed a selection for interacting with the antibody. This approach, using label selection affords high sensitivity and could be applicable for mapping antigen-antibody interaction site/s.

Adenovirus DNA polymerase is a phosphoprotein.

Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

Thyroglobulin glycosylation: location and nature of the N-linked oligosaccharide units in bovine thyroglobulin.

The cDNA sequence of bovine thyroglobulin (bTg) predicts 14 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of bovine thyroglobulin in order to establish actual glycosylation sites. The distribution of oligosaccharide types within the known sites of N-linked glycosylation has also been examined. Each glycopeptide was subjected to neutral and amino sugar analysis, as well as sialic acid analysis. Thirteen of the 14 putative N-linked glycosylation sites in bTg were confirmed as glycopeptides in the mature protein. Nine of these confirmed glycosylation sites appear to bear complex or hybrid type oligosaccharide units and four contain oligosaccharide structures in which only mannose and glucosamine were seen. Complex or hybrid type glycosylation was confirmed at asparagine residues 91, 464, 476, 834, 928, 1121, 1757, 1851, and 2232, while oligosaccharides containing only mannose and glucosamine were found at asparagine residues 1346, 1995, 2104, and 2277. Analysis of the amino acid sequence in the region of each putative glycosylation site predicts a high probability of a beta turn at 10 of the 13 sites. While glycosylation was distributed through most of the length of the thyroglobulin sequence, no oligosaccharides containing only mannose and glucosamine were found in the N-terminal half of the molecule. Several of the glycosylated peptides showed microheterogeneity and occurred in two or more discrete peaks on HPLC. A single putative site at asparagine residue 179 was not recovered in the purified glycopeptide population.

Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.

A neuropeptide Y (NPY)-related peptide is present in the river lamprey CNS.

Pancreatic polypeptide (PP)-like material from brain+spinal cord, and retina extracts of Lampetra fluviatilis was studied by HPLC and RIA. The brain+spinal cord extract showed a complex elution profile with multiple peaks of immunoreactivity. The retina extract showed a much simpler pattern with a single significant peak along with a trace of a second peak corresponding to the latest and penultimate peaks in the brain extract. Twenty-one out of 36 residues could be sequenced from the latest eluting peak in the brain extract. This sequence showed 81% identity with porcine neuropeptide (NPY) suggesting that both the brain/spinal cord and retina of the river lamprey contain a peptide homologous to NPY.

Placental prolactin-like protein A. Identification and characterization of two major glycoprotein species with antipeptide antibodies.

The purpose of this investigation was to develop immunologic probes to prolactin-like protein A (PLP-A) that could be used to characterize the protein and its distribution in various tissues. Five oligopeptides corresponding to different regions of the predicted PLP-A amino acid sequence (peptides 1-13, 62-76, 101-114, 129-145, and 152-164) were chemically synthesized by solid phase methodology. The peptides were purified to homogeneity by reverse phase high pressure liquid chromatography and coupled to keyhole limpet hemocyanin. The peptide-keyhole limpet hemocyanin conjugates were used to immunize rabbits. Immune responses were monitored by enzyme-linked immunoassay. Reactivity of the antipeptide antisera with placental proteins was determined by immunoblotting and immunoprecipitation analyses. All of the peptides except peptide 1-13 yielded significant immune responses. Antisera to peptides 101-114, 129-145, and 152-164 each specifically recognized proteins of Mr 29,000 and 33,000 from cytosol preparations of rat placental tissue and showed limited or no cross-reactivity with other members of the prolactin-growth hormone family. Three experiments were performed to determine whether the Mr 29,000 and 33,000 species were glycosylated derivatives of an Mr 25,000 precursor. Treatment of placental cytosolic preparations with N-Glycanase prior to immunoblotting resulted in the identification of only an Mr 25,000 species. It was also determined that the Mr 29,000 and 33,000 species specifically bound to concanavalin A. Furthermore, tunicamycin shifted the synthesis of PLP-A by placental explants from the Mr 29,000 and 33,000 forms to the Mr 25,000 species. The Mr 29,000 and 33,000 species were identified in serum obtained from pregnant and fetal rats but not in serum from nonpregnant females or males. We conclude that PLP-A is expressed in rat placenta. An Mr 25,000 precursor (predicted from PLP-A cDNA and these results) is glycosylated to either the Mr 29,000 or 33,000 form, both of which predominate in placenta and in circulation.

Hormone-containing peptides from normal and goiter human thyroglobulins.

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.

Structure of a porcine relaxin inactive on the mouse pubic ligament.

In addition to B31 (CM-a) and B28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B. Sequence analysis indicates that the two major components of relaxin C, like the other porcine relaxins, consist of two polypeptide chains linked by disulfide bonds. The shorter (A) chains are identical to the A chains of the other porcine relaxins, except for the absence of the N-terminal arginine residue. The B chains display microheterogeneity; the B sequences of the two predominant species are the same as those of the other porcine relaxins through B25, but terminate at valine residue B25 or serine residue B26, respectively.

An immunochemical study of avian pancreatic polypeptide: the nature of the principal epitope.

Structural features contributing to the antigenic recognition of the small globular hormone avian pancreatic polypeptide (APP) by a polyclonal antiserum have been defined using a solution phase radioimmunoassay technique. Cross-reactivity studies with PP homologues suggest that the surface residues within the alpha-helix of the peptide may be antigenic, whereas hydrophilicity and atomic mobility predictive methods implicate the molecules beta-turn region. Immunochemical data and circular dichroism measurements on a timed trypsin digest of APP indicate that the secondary structure of the alpha-helix is vital for the molecule's immunological competence. Immunoreactivities of iodinated derivatives of APP, as well as that of peptide fragments of APP and its homologues, support the importance of teritary structure involving the interaction of the polyproline and alpha helices. The highly mobile C-terminal residues 34-36 (His-Arg-Tyr-NH2) have been found by immunological analysis to be unimportant. Arginine residue 33, which has been conserved through vertebrate evolution, is a major antigenic contributor, since a large decrease in immunoreactivity, not accompanied by a significant change in conformation, was observed upon specific removal of this residue by carboxypeptidase B. These results are consistent with a "discontinuous" epitopic model for APP in which Arg-33 and exposed residues in the alpha-helix are principal components of an epitope or epitopes mediated by the secondary and tertiary structures of the molecule.

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides.

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.

A comparison of 30-kDa and 10-kDa hormone-containing fragments of bovine thyroglobulin.

Studies have been carried out on reduced and alkylated 19 S bovine thyroglobulin to characterize naturally occurring, iodine-rich fragments. In this report, the purification and properties of a 30-kDa, hormone-enriched polypeptide (TgE) are described and compared to that of a previously reported 10-kDa fragment (TgF). The amino acid sequence of TgF was found to overlap with that of TgE. In spite of its larger size, TgE contains only a single hormone bearing site. Both the 10- and 30-kDa fragments are derived from the NH2-terminal end of the bovine thyroglobulin. These fragments contain the principal hormone-forming site at residue 5 of the thyroglobulin sequence and appear to be formed by cleavage of the parent polypeptide chain. The mechanism which generates these cleavages is not clear since the sequences surrounding the cleavage points which give rise to these peptides are quite different. These two fragments may be precursor and product in such a process. The amino acid sequence contained within TgE includes two putative sites for N-linked glycosylation. Since no glucosamine was observed and only small amounts of neutral sugar were detected, it appears that this part of the molecule is not extensively glycosylated.

Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide.

Oxyntomodulin, glucagon, and a glucagon-like peptide (GLP) have been isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish. The three peptides were isolated by gel filtration and HPLC and were identified by size, composition, and glucagon-like immunoreactivity. The amino acid sequences of the oxyntomodulin and GLP were determined. The oxyntomodulin contains 36 amino acid residues and its sequence is H S Q G T F T N D Y S K Y L D T R R A Q D F V Q W L M S T K R S G G I T. The composition of the glucagon is identical to the N-terminal 29 residues of the gar oxyntomodulin. The single form of GLP found contains 34 amino acid residues in the following sequence: H A D G T Y T S D V S S Y L Q D Q A A K K F V T W L K Q G Q D R R E. These findings suggest that all three peptides are derived from a common precursor.

Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide.

Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV.

Digestion of thyroglobulin with purified thyroid lysosomes: preferential release of iodoamino acids.

[131I]Thyroglobulin [( 131I]Tg), prepared by either enzymatic iodination of human goiter Tg in vitro or isolation from the thyroids of rats previously injected with 131I, was digested with a solubilized enzyme mixture prepared from purified hog thyroid lysosomes. The digestion was performed at 37 C for 24 h under nitrogen at pH 5.0 in the presence of 4 mM dithiothreitol. Under these conditions the release of free [131I] iodoamino acids (MIT, DIT, T4, and T3) was quantitatively very similar to that observed with a standard pronase digestion procedure. To determine whether other amino acids in Tg were released as quantitatively as the iodoamino acids, free amino acids in the lysosomal digest were measured, and total free amino acid release was compared with a similar analysis performed after digestion of [131I]Tg with 6 N HCl. Total amino acid release was much less complete than iodoamino acid release, indicating preferential release of iodoamino acids from Tg by lysosomal digestion. Analysis of the lysosomal digest by HPLC on a size exclusion column indicated that Tg was degraded to peptides with a mol wt less than 4000. Assuming that the in vitro lysosomal digestion system represents a valid model for the physiological proteolytic system that degrades Tg, the results of the present study suggest that a substantial portion of the Tg in the thyroid is not degraded to free amino acids and that peptide fragments of Tg are normally present in the thyroid. In such a case, the fate and possible physiological activity of these fragments require further elucidation.

Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y.

The amino acid sequence of a peptide isolated from the Pacific salmon (Oncorhynchus kisutch) endocrine pancreas has been determined. This simple 36 residue peptide is a member of the pancreatic polypeptide family. It contains a C-terminal tyrosinamide and is more homologous with porcine neuropeptide Y (NPY) (83%) and peptide YY (75%) than any of the previously characterized pancreatic polypeptides (PP). This peptide appears to be the major but not the only representative of this family of peptides present in the endocrine pancreas of this fish. This peptide is referred to as salmon pancreatic polypeptide (salmon PP).

The Cuisinart food processor efficiently disaggregates tissues.

The Cuisinart Model DLC-7 PRO food processor efficiently disaggregates plant and animal tissues resulting in time savings and often increased yields in the isolated material.