Glen L. Hortin

High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of polypeptide abundance.

BACKGROUND Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass. CONTENT We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 micromol/L on a molar basis or from about 50,000 to 1 mg/L mass abundance. CONCLUSIONS Data on the concentrations of the high-abundance peptide chains in plasma assist in understanding the composition of plasma and potential approaches for clinical laboratory or proteomic analysis of plasma proteins. Development of more extensive databases regarding the plasma concentrations of proteins in health and diseases would promote diagnostic and proteomic advances.

Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence

A wide variety of secretory proteins have recently been found to undergo post-translational sulfation of specific tyrosine residues. Here, amino acid sequences surrounding known sulfation sites in proteins are analyzed in order to identify factors which determine the specificity of sulfation. Several distinctive features of sulfation sites are identified, including: abundance of acidic amino acid residues, lack of basic residues, low hydropathy, absence of neighboring cysteine residues, lack of extended secondary structure. Rules are proposed for predicting likely sites of sulfation based on the amino acid sequence of a protein.

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: Application of jacalin-agarose

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.

Differences in Values Obtained with 2 Assays of Prostate Specific Antigen

Several different assays for prostate specific antigen have become available for use in clinical laboratories. We compared the 2 most widely used assays, the Tandem-R and the Pros-Check prostate specific antigen assays, to determine if different assays yield comparable results. We analyzed 70 serum specimens from patients with suspected or known prostatic carcinoma by both assays. Results from the assays showed close linear correlation (r equals 0.988) but the Pros-Check assay yielded values 1.85 times those of the Tandem-R test. The proportional bias between assays is owing to differences in the values assigned to calibration standards in the assays, and it demonstrates a need for improved standardization of prostate specific antigen assays. Important consequences of the bias between assays are that different normal ranges apply and that values from the 2 assays cannot be compared directly.

Diagnostic potential for urinary proteomics

Urine represents a modified ultrafiltrate of plasma, with protein concentrations typically approximately 1000-fold lower than plasma. Urines low protein concentration might suggest it to be a less promising diagnostic specimen than plasma. However, urine can be obtained noninvasively and tests of many urinary proteins are well-established in clinical practice. Proteomic technologies expand opportunities to analyze urinary proteins, identifying more than 1000 proteins and peptides in urine. Urine offers a sampling of most plasma proteins, with increased proportions of low-molecular-weight protein and peptide components. Urine also offers enriched sampling of proteins released along the urinary tract. Although urine presents some challenges as a diagnostic specimen, its diverse range of potential markers offers great potential for diagnosis of both systemic and kidney diseases. Examples of clinical situations where this may be of value are for more sensitive detection of kidney transplant rejection or of renal toxicity of medications.

Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose

Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.

α2-antiplasmin's carboxy-terminal lysine residue is a major site of interaction with plasmin

alpha 2-Antiplasmin (AP) inhibits plasmin in a two-step reaction in which AP reversibly binds to lysine-binding sites of plasmin and, then, more slowly complexes covalently with the enzymes active site. Here, we show that the C-terminal lysine residue of AP has a key role in binding of the inhibitor to plasmin. A synthetic peptide corresponding to the C-terminal 26 amino acid residues of AP blocked association of AP with plasmin, but this activity of the peptide was lost when its C-terminal lysine residue was removed with carboxypeptidase B. The essential role of this lysine residue was shown more directly by treating AP with carboxypeptidase B and observing that AP lost its ability to inhibit plasmin rapidly.

[61] Applications of amino acid analogs for studying co- and posttranslational modifications of proteins

Publisher Summary This chapter presents general considerations of applications of amino acid analogs for studying co- and post-translational modifications of proteins. This approach involves selection of amino acid analogs, selection of analog concentration, demonstration of amino acid analog incorporation into protein, and analysis of the effects of amino acid analogs on protein processing. When a particular analog is applied to a new system, it is necessary to assess critically whether the analog is incorporated into protein and to establish its effective concentration. Selection of amino acids for incorporation into protein depends on the selectivity of aminoacyl-tRNA synthetases, which possess an extraordinary ability to discriminate among structurally similar amino acid substrates. However, determination of the appropriate analog concentration for use with intact cells presents greater difficulty owing to the added complications of uptake into cells, amino acid metabolism, and endogenous amino acid pools.

Miscleavage at the presequence of rat preprolactin synthesized in pituitary cells incubated with a threonine analog

We have shown previously that processing of preprolactin to prolactin in isolated rat pituitaries is inhibited by the threonine analog, beta-hydroxynorvaline (Hnv), presumably because of its substitution for the threonine at the cleavage site. Here, we show by amino-terminal sequence analyses that Hnv altered the site at which preprolactin produced had three extra amino acids at the amino terminus. The miscleaved prolactin was secreted into the medium, and there was a delay of approximately 5 min between the initial appearance of prolactin and the formation of miscleaved prolactin. This lag period suggests that miscleaved prolactin did not represent an intermediate in the normal processing of preprolactin but resulted from cleavage of completed preprolactin chains containing Hnv. These results show that modification of the structure of a preprotein can alter the site of its cleavage. One site that appears to be critical for correct cleavage is the final amino acid residue in the prepeptide. The data also indicate that complete removal of the presequence is not required for secretion of a protein.

Inhibitors of the sulfation of proteins, glycoproteins, and proteoglycans

Two categories of compounds, substrates of sulfation and sulfate analogs, were tested for the ability to inhibit sulfation of macromolecules secreted by HepG2 cells. Several compounds which most effectively inhibited sulfation without toxic effects on cells were tested for their relative inhibition of sulfation of tyrosine residues (using the fourth component of complement as a model substrate), of N-linked oligosaccharides (alpha 2HS-glycoprotein as substrate), and of proteoglycans. Inhibitors decreased the sulfation of all three classes of substrate, but not always equally. Use of inhibitors from both categories in combination yielded synergistic effects, with more effective inhibition of sulfation and low toxicity. Such combinations of inhibitors should provide a valuable tool for probing the significance of the sulfation of macromolecules.