Sites Asn Research Articles

CDNA sequence and tissue expression analysis of glucokinase from liver of grass carp (Ctenopharyngodon idella)

A full-length cDNA coding glucokinase (GK) was cloned from the liver of grass carp ( Ctenopharyngodon idella ) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The cDNA obtained was 2066 bp exclusive of poly (A) residues with a 1431 bp open reading frame encoding 476 amino acids. The GK protein has a calculated molecular weight of 53.7 kDa and isoelectric point of 5.11. Some conserved functional sites were found including one conserved hexokinase signature sequence Leu 156 -Phe 181 ; two N-linked glycosylation sites Asn 176 and Asn 214 ; one cell attachment sequence Arg 202 -Asp 204 ; one glycosaminoglycan attachment site Ser 455 -Gly 458 . The amino acid sequence has a high similarity to GK of other species, the percent identity compared with topmouth culter, common carp, human and rat are 98.1, 96.8, 80.3 and 79.8%, respectively. Tissue distribution of GK mRNA in brain, mesenteric adipose tissue, spleen, white muscle and liver of grass carp was analyzed by SYBR real-time fluorescence quantitative RT-PCR method using β-actin as an internal control for cDNA normalization. The result shows that the expression level of GK mRNA in liver was significantly higher than in mesenteric adipose tissue, spleen and brain (p<0.05). Relative expression profile of GK mRNA in liver normalized with β-actin level was 31, 454 and 649-fold compared with the levels in mesenteric adipose tissue, spleen and brain, respectively. Meanwhile, GK mRNA was not detected in white muscle. Key words : Ctenopharyngodon idella , glucokinase, full-length cDNA, tissue distribution, real-time PCR.

The Alg5 ortholog Wollknäuel is essential for correct epidermal differentiation during Drosophila late embryogenesis

The formation of an extracellular matrix (ECM) presupposes an ordered delivery of its components to ensure its stereotypic architecture. The Drosophila cuticle is an ECM produced by the epidermis at its apical site and is characterized by a layered organization. To understand the mechanisms of cuticle assembly during development, we have investigated early aspects of protein N-glycosylation, i.e. the attachment of a dolichol-linked oligosaccharide to distinct Asn sites of a protein known to be essential for sorting in the secretory pathway. Mutations in the Drosophila alg5 gene wollknäuel (wol) that codes for an enzyme initiating the glucosylation of the dolichol-linked oligosaccharide decrease, as expected, glucosylation and the amounts of N-glycosylated proteins such as the cuticle-organizing factor Knickkopf, without affecting their correct localization. At the same time, the polarity determinants Crumbs and atypical protein kinase C accumulate at the apical plasma membrane in wol deficient embryos. In part, these perturbations may also be caused by the unfolded protein response, which is commonly triggered by ER stress and downsizes transcription and translation in general. In any case, they are associated with the loss of cuticle layering and aberrant apical plasma membrane organization suggesting that glucosylation, either directly or indirectly through controlling protein degradation, is important for the efficient and balanced deployment of the biochemical functions of secreted and membrane-associated proteins during epidermal differentiation.

CDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (Cyprinus carpio Var. Jian)

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24-354 residues) and C-terminus (355-507 residues). Before N-terminus, 1-23 residues is signal peptide, 6-23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn(41) and Asn(88); one catalytic triad Ser(174), Asp(198) and His(283); one conserved heparin-binding site Arg(321) to Arg(324) (RKNR); eight cysteines residues Cys(69) and Cys(82), Cys(258) and Cys(281), Cys(306) and Cys(325), Cys(317) and Cys(320) which are involved in four disulfide bridges; one polypeptide "lid" that participates in substrate specificity. At C-terminus, Asn(401) is another N-linked glycosylation site, and Trp(434) and Trp(435) (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using beta-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.

Prediction of glycosylation sites using random forests

BackgroundPost translational modifications (PTMs) occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function.ResultsWe use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program), predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types.ConclusionWe have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at .

Selection Against Glycosylation in Ruminant Pancreatic Ribonucleases by Replacements in the Ancestral Carbohydrate Attachment Site

A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn–Met–Thr at residues 34–36, was experimentally confirmed. The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.

2-Deoxy-d-glucose as a potential drug against fusogenic viruses including HIV

Most enveloped viruses fuse with host cells and catalyze fusion among host cells by expression of specific patterns of N-glycosylation on their envelope proteins. In the 1970s, it was observed that 2-deoxy-D-glucose (2DOG) and 2-fluoro-2-deoxy-D-mannose (2F2DOM) inhibited N-glycosylation of asparagine (Asn) sites on the external domain of viral envelope proteins. This effect led to the virus particles being non-fusogenic with greatly reduced infectivity and reduced ability to pass from cell to cell by catalyzing cell--cell fusion. At that time, this observation was not particularly important because viral diseases were readily prevented by vaccines and there was no known link between fusogenic viruses and cancer. Today, we are faced with a chronic and lethal viral disease (AIDS) caused by a virus (HIV) that mutates so quickly that we have not been able to produce a vaccine. Moreover, it is spreading among millions of people unable to afford more than basic medications. In addition, cell--cell fusion has been identified as an important, if not essential, step in the progression of abnormal cell clones to clinically significant cancer and fusogenic viruses have been shown to cause progression of some tumors. Here, we reiterate the hypothesize (first made in 1986 by Blough et al. [Blough HA, Pauwels R, De Clercq E, Cogniaux J, Sprecher-Goldberger S, Thiry L. Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins. Biochem Biophys Res Commun 1986; 141:33-8]) that 2DOG, 2F2DOM and related compounds, which interfere with normal N-glycosylation of virus envelope proteins, are attractive candidates for anti-fusogenic drugs that can be used against chronic virus diseases and cancers. This analysis also supports the concept of blocking N-acetylglucosaminyl-transferases with chloroquine or other drugs (proposed by Savarino et al. [Savarino A, Lucia MB, Rastrelli E et al. Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors. J Acquir Immune Defic Syndr 2004; 35:223-32]) as an anti-viral approach. These drugs may have broader utility and lower cost than drugs designed specifically to target the gp41 protein of HIV, which have become popular as viral-entry inhibitors for treatment of HIV/AIDS.

Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen

Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.

Protein L-Isoaspartyl Methyltransferase Catalyzes in Vivo Racemization of Aspartate-25 in Mammalian Histone H2B

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V.

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu 2+ (from 1 to 750 μg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74–85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn 23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.

Glycosylation of the calcitonin receptor-like receptor at Asn 60 or Asn 112 is important for cell surface expression

The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn 60, Asn 112 and Asn 117 are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [ 125I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn 117, and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn 117 by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.

Post-translational processing of two α-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the α-amylase inhibitors αAI-1 and αAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in αAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that αAI-1 and αAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the α 1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.

Cloning of mouse gamma-glutamyl hydrolase in the form of two cDNA variants with different 5' ends and encoding alternate leader peptide sequences.

Mouse-liver gamma-glutamyl hydrolase (GH) is a lysosomal endopeptidase with an acid pH optimum that is activated by sulfhydryl compounds and preferentially hydrolyzes the most proximal gamma-glutamyl linkage of longer chain polyglutamates of folates and their analogues. We describe the cloning of this mouse lysosomal cDNA enzyme from liver GH mRNA in the form of two cDNA variants (1.295 and 1.268 kb in length) differing 14-fold (Variant I versus Variant II) in relative frequency that exhibited 5'-end heterogeneity and encoded alternate leader peptides. The 5' UTR in these variants also differs in length by 27 nucleotides. Otherwise, the ORF and 3' UTR in each case are the same. These cDNAs encode a protein in which the deduced amino acid sequence shares 78.9 and 69. 1% identity to rat and human GH sequences, respectively. Amino acid sequence comparisons among the three species identified three conserved Asn sites and two conserved Cys residues that may be sites of glycosylation and sulfhydryl compound activation, respectively. Variant I GH mRNA was more abundant than Variant II GH mRNA in all mouse tissues examined. Variant I GH mRNA levels were extremely high in salivary gland, moderately high in kidney, liver, lung, stomach and uterus, low in small intestine, brain and fetal liver and relatively rare in thymus, spleen and skeletal muscle. Abundance of GH mRNA among tumors varied from low to high, with no discernible correlation with their tissue of origin.

Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme.

Lipoprotein lipase (LPL) and hepatic lipase (HL) share two conserved asparagine-linked glycosylation sites, located at the amino- and carboxy-terminal domains of the protein. Human HL contains two additional sites, preceding each conserved site by 36 and 35 amino acids, respectively. The utilization of these sites for glycan-binding and the role of each glycan chain for the catalytic function of human LPL, rat HL, and human HL was investigated. To accomplish this aim, potential Asn glycosylation sites were changed to Gln by site-directed mutagenesis and the resulting constructs were expressed in a mammalian (COS) cell system. We demonstrate the following. 1) All potential glycosylation sites in human LPL, rat HL, and human HL are utilized. 2) Lack of glycosylation at the two non-conserved sites in human HL has no effect on enzyme expression. 3) Glycosylation at the conserved Asn sites in the N-terminal domain of LPL and HL is required for the synthesis of a fully active and secreted lipase. While this is an absolute requirement for LPL, a portion (approximately 25%) of HL molecules lacking glycosylation at this essential site still becomes active and secreted. However, the simultaneous elimination of both glycosylation sites at the N-terminal domain of human HL results in the virtual abolishment of enzymatic activity and secretion. 4) Glycosylation at the conserved sites in the C-terminal domain is not essential for the expression of active lipases. 5) Eliminating all glycosylation sites in LPL and HL results in the synthesis of inactive enzymes that are retained intracellularly; however, a small portion (2%) of unglycosylated rat HL was active and secreted. We conclude that glycosylation overall plays an important role in the formation of functional LPL and HL.

Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator.

When incubated at pH 7.3, 37 degrees C, human recombinant tissue plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.

N5-Methylasparagine and asparagine as nucleophiles in peptides: main-chain vs. side-chain amide cleavage

The chemistry of peptides containing N 5 -methylasparagine (NMA) was investigated by incubating the synthetic peptides Ile-Ala-Pro-Gly-Gly-Asn-Gly-Tyr and Ile-Ala-Pro-Gly-Gly-NMA-Gly-Tyr at 60 o C in 0.1 M NaPO 4 , PH 7.4, to assay for peptide deamidation. The Asn-Gly octapeptide deamidates to Ile-Ala-Pro-Gly-Gly-isoAsp/Asp-Gly-Tyr with a half-life of 2.17 h and activation energy of 18.6 kcal/mol. The NMA-Gly octapeptide partitions between main-chain cleavage and side-chain deamidation in 2.7:1 ratio. Analysis of products diagnostic for each of these NMA peptide reactions yields indistinguishable activation energies for each pathway: 22.6 kcal/mol. The half-life for NMA side-chain deamidation is 98h, commensurate with a 2.5 kcal/mol difference in activation free energies for deamidation at Asn and NMA sites. These results indicate that methylation provides a substantial (45-fold) stabilization against intramolecular C-N cleavage. The identical activation energy for the alternative pathways of NMA peptide reactivity suggests the differences in the rates may be due to the preexponential portion of the rate equation reflecting small differences in ΔS * . Molecular mechanics studies were performed to account for these patterns. The computational studies disclose 3-fold more conformers in the Boltzmann population for the tetrahedral intermediate leading toward main-chain cleavage. This result supports the hypothesis that the 2.7-fold difference in NMA peptide partitioning rates is attributable to differences in ΔS *

Conversion of the human 5-HT 1Dβ serotonin receptor to the rat 5-HT 1b ligand-binding phenotype by Thr 355 ASN site directed mutagenesis

The human 5-HT 1Dß serotonin receptor and its rat homolog (also called the 5-HT 1B receptor) share 93% amino acid identity, yet display markedly different pharmacological specificities. Comparison of deduced amino acid sequences among these and other recently cloned receptors suggested that this phenotypic difference might be attributable to a single human threonine 355/rat asparagine 351 amino acid difference in the putative seventh membrane spanning regions. We now report that Thr 355Asn mutagenesis of the human S-HT 1Dß receptor alters the binding characteristics of the recombinant receptor in [ 3H]5-HT binding assays to a profile very similar to that of the rat S-HT 1B binding site. These results confirm that this single amino acid difference is responsible for the majority of the known pharmacological discrepancies between human and rat observed for 5-HT 1Dß (S-HT 1B) receptors.

N-glycosylation site mapping of human serotransferrinby serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy

This report describes the N-glycosylation site mappingof human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi- α(2→6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn 413 and Asn 611) in the ratio of ∼85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl2,4-triantennary and a trisialyl2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn 413 was found to be ∼5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asnell in the ratio ∼1:1.

Amino acid sequence of human acidic fibroblast growth factor

The complete amino acid sequence of human brain acidic fibroblast growth factor (aFGF) has been established. Human aFGF consists of 140 amino acids and is highly homologous to bovine aFGF (11 amino acid replacements). Results from experiments involving alkylation of cysteine residues are compatible with the possibilities that in aFGF all three cysteines exist as free sylfhydryls, or alternatively, that a disulfide bridge is present but cannot be identified due to disulfide scrambling caused by the SH group of the remaining cysteine. A potential glycosylation site Asn 114-Gly 115-Ser 116 is present in aFGF but the mitogen does not bind to lectins suggesting that it may not be glycosylated.