Isolation and characterization of the major gel proteins in human semen, semenogelin I and semenogelin II.

Semenogelin I and semenogelin II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubilized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/l urea and dithiothreitol. This protected the semenogelins from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatography on heparin-Sepharose. Semenogelins I and II were almost selectively retained and eluted partially separated in 0.25 mol/l NaCl. Further purification was achieved by chromatography on Superose. Approximately 10-20 mg semenogelin I and 2-5 mg semenogelin II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogelin I (49 958 Da) and the major form of semenogelin II (63 539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogelin II was due to asparagine-linked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate amino-peptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenogelins I and II were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.

Plasma protein changes induced by two orally administered androgen derivatives.

Epostane is a synthetic 17 alpha-alkylated 5 beta-androstane derivative, active following oral administration and devoid of any apparent androgenic, estrogenic or antiestrogenic potency. Circulating concentrations of 13 different plasma proteins were measured in eight women before and after 2 and 4 weeks of daily oral intake of 600 mg of epostane. The results were compared with those previously found during administration of the same daily dose of danazol, a synthetic 17 alpha-alkylated androgen derivative with known androgenic/anabolic activity. Epostane significantly suppressed serum levels of sex hormone-binding globulin, pregnancy zone protein and thyroxin-binding globulin and increased the levels of transthyretin. Haptoglobins, plasminogen and transferrin showed minor and/or transient changes and the levels of high density lipoproteins, alpha2-macroglobulin, albumin, C1-esterase inactivator, C3 complement and transcortin remained unaffected. The pattern of changes in plasma proteins was almost identical to that induced by administration of danazol, although the effects of epostane were somewhat weaker. Thus epostane is capable of inducing substantial changes in the pattern of steroid-sensitive plasma proteins in an androgen-like fashion despite its apparent lack of androgenic activity. The capacity of a steroid to induce such changes thus seems to be tied to the chemical structure rather than to the intrinsic hormonal activity of the molecule.

Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp-->Asn).

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63-->Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62-->Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.

The pregnancy zone protein response during gestation: a metabolic challenge.

Prospective studies of pregnant women were performed to compare individual variations in the plasma concentration of sex hormone binding globulin (SHBG) and pregnancy zone protein (PZP) during pregnancy, and to elucidate the degree of co-variation between these oestrogen sensitive proteins during gestation. The plasma concentration of SHBG manifested continuous increase reaching a 12-fold peak at delivery. The increase of the protease inhibitor PZP paralleled that of SHBG reaching a peak with a 25-fold increase by the beginning of the third trimester. Then it started to decline, while that of SHBG continued to increase. The synthesis of the protease inhibitor may also continue to increase during late gestation but its elimination from the circulation may be accelerated when the syncytiotrophoblastic area in contact with the maternal blood approaches its maximum. The unusually wide individual variation of PZP concentrations in non-pregnant women was confirmed. However, the individual levels increased proportionally during the progress of pregnancy, and we may speak of low, medium and high reactors for PZP. One initial conclusion to be drawn from the present findings is that the value of the plasma PZP concentration can only be interpreted from a pathophysiologic point of view if the patient's baseline level is known.

Alloalbuminemia in Sweden: structural study and phenotypic distribution of nine albumin variants.

Plasma samples exhibiting alloalbuminemia on electrophoresis at pH 8.6 were requested from clinical laboratories throughout Sweden. Nine variants, each representing a different single point mutation, were found in 100 apparently unrelated Swedes. The overall prevalence of alloalbuminemia was estimated at 1:1700. Mutations were identified by protein-structural analysis followed by allele-specific DNA hybridization to verify the most common types. Slightly retarded (+1) mobility was seen in 80 cases. Of these, 71 had the Arg(-2)----Cys proalbumin variant previously called Malmö I proalbumin. Thirteen examples of the second most frequent type, the substitution Lys313----Asn and a mobility change of -1 charge unit, were found, as well as six cases of Glu570----Lys (albumin B) and a single case of Arg-1----Gln (proalbumin Christchurch). Five previously unreported types of alloalbuminemia were identified: four instances of Glu376----Gln, which is the second known mutation at this site; two examples of Asp550----Ala, the second mutation reported at this site; and one example each of Asp63----Asn, Gln268----Arg, and Asn318----Lys. Other mutations were identified among eight subjects of foreign descent. The high frequency and relatively uniform geographic distribution of the Arg-2----Cys mutation suggest that it may have occurred in a founder individual many generation ago in Sweden.

Plasma protein homeostasis in chronic hemodialysis patients.

The concentrations of 25 plasma proteins were measured in 29 patients with chronic renal insufficiency. All the patients had terminal renal failure and were treated with intermittent hemodialysis, but were otherwise in good general condition at the time of investigation. The plasma levels of 8 proteins with M(r) < 50 kD were significantly elevated compared to normal subjects. In contrast, only 2/17 proteins of greater size were found in increased concentrations. The degree of increase in concentration differed substantially between individual low molecular weight proteins, suggesting a complex metabolism in addition to delayed renal elimination. Acute phase proteins and immunoglobulins were not affected by renal insufficiency per se, although erythrocyte sedimentation rates were generally high. The synthesis of acute phase proteins increased normally during the course of inflammation. We conclude that although the sedimentation rate is of no value, complicating inflammatory processes can be traced by quantitative analysis of acute phase proteins, including C-reactive protein, even in patients with severe chronic renal insufficiency.

Crystal structure of cleaved human alpha 1-antichymotrypsin at 2.7 A resolution and its comparison with other serpins.

The crystal structure of proteolytically modified human alpha 1-antichymotrypsin (ACT), a member of the serpin superfamily, has been solved by Paterson search techniques and refined to an R-factor of 18.0% at 2.7 A resolution with mean deviations from standard bond lengths and angles of 0.013 A and 3.1 degrees, respectively. The final model consists of 374 amino acid residues, 126 solvent molecules and five sugar residues. Asn70 could be identified unambiguously as a glycosylation site and Asn104 is probably also glycosylated. The structure of cleaved ACT is compared with cleaved alpha 1-antitrypsin (alpha 1 PI) and with plakalbumin, which are prototypical models for cleaved and intact serpins, respectively. Cleaved ACT is very similar to cleaved alpha 1 PI; in particular, it has strand s4A, which is liberated by proteolysis, inserted as the middle strand in beta-sheet A. ACT and alpha 1 PI differ locally only at sites of insertions, except at the segment s3C-turn-s4C, which is displaced by several angström units. This region of ACT is involved in DNA binding.

Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors.

Prostate-specific antigen (PSA) is one of the three most abundant prostatic-secreted proteins in human semen. It is a serine proteinase that, in its primary structure, manifests extensive similarities with that of the Arg-restricted glandular kallikrein-like proteinases. When isolated from semen by the addition of chromatography on aprotinin-Sepharose to a previously described procedure, PSA displayed chymotrypsin-like activity and cleaved semenogelin and the semenogelin-related proteins in a rapid and characteristic pattern, but had no trypsin-like activity. About one third of the purified protein was found to be enzymatically inactive, due to cleavage carboxy-terminal of Lys145. Active PSA formed SDS-stable complexes with alpha 1-antichymotrypsin, alpha 2-macroglobulin-analogue pregnancy zone protein. PSA formed inhibitory complexes with alpha 1-antichymotrypsin at a molar ratio of 1:1, a reaction in which PSA cleaved the inhibitor in a position identical to that reported from the reaction between chymotrypsin and alpha 1-antichymotrypsin. The formation of stable complexes between PSA and alpha 1-antichymotrypsin occurred at a much slower rate than that between chymotrypsin and alpha 1-antichymotrypsin, and at a similar or slightly slower rate than that between PSA and alpha 2-macroglobulin. When added to normal blood plasma in vitro, active PSA formed stable complexes both with alpha 2-macroglobulin and alpha 1-antichymotrypsin. This complex formation may be a crucial determinant of the turnover of active PSA in intercellular fluid or blood plasma in vivo.

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A.

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [Löbermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.

Hypermutability of CpG dinucleotides in the propeptide-encoding sequence of the human albumin gene.

An electrophoretically slow albumin variant was detected with a phenotype frequency of about 1:1000 in Sweden and was also found in a family of Scottish descent from Kaikoura, New Zealand, and in five families in Tradate, Italy. Structural study established that the major variant component was arginyl-albumin, in which arginine at the -1 position of the propeptide is still attached to the processed albumin. A minor component with the amino-terminal sequence of proalbumin was also present as 3-6% of the total albumin. After amplification of the gene segment encoding the prepro sequence of albumin, specific hybridization of DNA to an oligonucleotide probe encoding cysteine at position -2 indicated the mutation of arginine at the -2 position to cysteine (-2 Arg----Cys). This produced the propeptide sequence Arg-Gly-Val-Phe-Cys-Arg. This was confirmed by sequence analysis after pyridylethylation of the cysteine. This mutation produces an alternate signal peptidase cleavage site in the variant proalbumin precursor of arginyl-albumin giving rise to two possible products, arginyl-albumin and the variant proalbumin. Another plasma from Bremen had an alloalbumin with a previously described substitution (1 Asp----Val), which also affects propeptide cleavage. Hypermutability of two CpG dinucleotides in the codons for the diarginyl sequence may account for the frequency of mutations in the propeptide. Mutation at these two sites results in a series of recurrent proalbumin variants that have arisen independently in diverse populations.

The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism.

The S variant of the human alpha 1-antitrypsin with E-264----V, is responsible for a mild alpha 1-antitrypsin deficiency quite common in the European population. S protein specifically cleaved at the susceptible peptide bond was crystallized and its crystal structure determined and refined to 3.1 A resolution. The S variant crystallizes isomorphous to the normal M variant. The difference Fourier electron density map shows the E----V change as outstanding residual density. In addition, small structural changes of the main polypeptide chain radiate from the site of mutation and affect parts far removed from it. By the mutation, internal hydrogen bonds and salt linkages of E-264 to Y-38 and K-487, respectively, are lost. They cause the far-reaching slight distortions and are probably related to the reduced thermal stability of the S mutant. They may also be responsible for slower folding of the polypeptide chain and the clinical symptoms of alpha 1-antitrypsin deficiency. In a theoretical study by molecular dynamics methods simulations of the M and S proteins were made and the results analysed with respect to structural and dynamic properties and compared with the experimental results. There is a significant correlation between experimental and theoretical results in some respects.

The amino acid substitutions of human alpha 1-antitrypsin M3, X and Z.

alpha 1-Antitrypsin has been isolated from individuals with inherited genetic variants M3, X and Z. A fragmentation and peptide mapping system is described which together with amino acid and sequence analyses revealed the substitutions in M3 at 376 of Glu to Asp, in X at 204 of Glu to Lys and in the physiologically innocent Z a mutation at 213 of Val to Ala. The latter represents a second amino acid substitution in the Z protein.

Sequential changes of the plasma-protein pattern in cases of hepatitis A.

The level of 21 plasma proteins was followed in Hepatitis A for two months after onset of icterus. The mean concentration of alpha 1-antitrypsin, orosomucoid, haptoglobin, C-reactive protein (CRP) and alpha 1-antichymotrypsin increased uniformely during the first week of hepatitis A. Thus, they differ from that of inoculation hepatitis earlier described. The mean curve for IgM was higher in hepatitis A than the corresponding results for inoculation hepatitis during the first week of illness, but because of great inter-individual differences in concentrations IgM determinations can not be used to discriminate between the two diseases in a given case. IgA levels were slightly increased early in hepatitis A but no change in IgG levels was observed. Prealbumin was the best mirror of the patients' recovery or deterioration.

Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen.

The comparison of measurements of fibronectin and lactoferrin in ejaculates from vasectomized men, subjects with functional deficiency or aplasia of the seminal vesicles, and reference subjects provided evidence that both the fibronectin and the lactoferrin in human seminal fluid originate from the seminal vesicles and the ampullae. The fibronectin is incorporated in the framework of the seminal gel formed during the immediate postejaculatory phase, whereas the lactoferrin remains in solution. In the seminal gel fibronectin is linked to its predominant structural protein, a high molecular weight seminal vesicle protein (semenogelin). Both the gel-bound fibronectin and semenogelin are progressively fragmented and solubilized by the abundant prostatic kallikrein-like protease (prostate-specific antigen) during and after seminal gel liquefaction. Lactoferrin remains essentially unaffected by the seminal proteases.

The predominant protein in human seminal coagulate.

The predominant protein in human seminal vesicle secretion constitutes the structural protein of coagulated semen. This high molecular weight protein (HMW-SV-protein) is stable in seminal vesicle secretion during in vitro storage at 37 degrees C for at least 20 h, but is rapidly cleaved on mixing with prostatic proteases. Seminal coagulate, washed free of soluble components, is dissoluble by 2 to 3 mol/l of guanidine-HCl. Although dithiothreitol added to seminal coagulate does not liquefy the clot, complexes between HMW-SV-proteins are broken up by reduction under denaturing conditions, which suggests that the non-covalent linkages of HMW-SV-proteins are essential in the clot. Prostatic proteases cleave the HMW-SV-protein during liquefaction of ejaculated semen to a series of labile proteins. These proteins are further cleaved to peptides of successively decreasing size after completed liquefaction. The cleavage of the HMW-SV-protein is the major cause of the fast shift of the electrophoretic pattern of seminal proteins if semen is stored without protease inhibitors.

Characterization of the predominant basic protein in human seminal plasma, one cleavage product of the major seminal vesicle protein.

From liquefied human seminal plasma, we purified the predominant basic protein which appears following liquefaction of coagulated semen. The protein was purified in the presence of di-isopropylfluorophosphate to retard its degradation. Heparin-Sepharose chromatography was followed by gel filtration (Biogel P 60) and by fast performance liquid chromatography on a reversed phase column (C8). The basic protein is a single polypeptide chain with an apparent molecular mass of 12.8 kDa, and has a pI value between that of trypsinogen (9.3) and cytochrome C (10.3). The protein contains no carbohydrate, is rich in histidine, glutamate, and lysine, but is devoid of both cysteine and methionine. The amino-terminal portion of the protein sequence is unique: H N K Q E G R D H D K S K G H F H R V V I H H K G G K A H R G-. A specific rabbit antiserum was raised against the 12.8 kDa basic protein. The protein was found to be unique to seminal plasma among all extracellular fluids examined. Three immunologically related 52 kDa, 71 kDa, and 76 kDa proteins were identified in seminal vesicle secretion when it had been reduced. Prostatic enzyme(s) degraded these proteins to the 12.8 kDa basic protein and several other basic proteins with apparent molecular masses below 18 kDa.

Liquefaction of coagulated human semen.

Liquefaction of coagulated human semen was inhibited by o-phenanthroline; subsequent addition of Zn2+ reversed this inhibition, but not if the coagulum was repeatedly washed before Zn2+ was added. No liquefaction of the coagulum occurred when Fe2+ was added (in a 1:3 molar ratio to o-phenanthroline), and the gel repeatedly washed. This o-phenanthroline-depleted coagulum was liquefied by resuspended pellet from ultracentrifuged pooled seminal plasma. In denatured and reduced semen coagulate, we identified the 52 kDa, 71 kDa, and 76 kDa protein bands of the predominant seminal vesicle protein. The protein was not present in the supernatant after centrifugation of coagulated semen, and it was degraded to several minor basic proteins when semen liquefied. These findings imply that the predominant seminal vesicle protein functions as the structural protein of coagulated semen. In much the same way as in ejaculated semen, the 52 kDa, 71 kDa, and 76 kDa protein bands in seminal vesicle secretion collected postmortem were digested to minor basic proteins after incubating the secretion with resuspended pellet from ultracentrifuged seminal plasma. This pellet contained the membrane-bound succinyl(alanine)3-p-nitroanilide hydrolysing peptidase of prostatic origin which, like the liquefaction process, was active in the presence of EGTA, inhibited by non-chelated Zn2+, and inactivated by o-phenanthroline--an inactivation that was reversed by Zn2+.

The use of thiol-disulphide exchange chromatography for the automated isolation of alpha 1-antitrypsin and other plasma proteins with reactive thiol groups.

A method has been developed for the rapid isolation of alpha 1-antitrypsin and other thiol proteins from plasma by an automated chromatography system. The thiol-proteins are initially bound to matrix-linked activated thiol-compounds by an SH-SS interchange reaction. The mixed disulphides are then reduced in two steps and subfractionated by passage through Blue-Sepharose and AH-Sepharose columns. The rate of the interchange reactions varies with the microenvironment of the reacting thiols. alpha 1-Antitrypsin is recovered with 95% purity in 60% yield within two days from 1 l of plasma.

Screening for fetal malformations using ultrasound and measurements of alpha-fetoprotein in maternal serum.

Fetal ultrasound combined with semiquantitative measurements of alpha-fetoprotein in maternal serum was used for early detection of neural tube defects and omphalocele in 10 147 pregnancies. The accurate assessment of gestational age, obtained by ultrasound, facilitated evaluation of alpha-fetoprotein concentrations in selecting cases for amniocentesis. The advantage of screening with two independent methods is suggested by the finding that eight out of 10 cases with malformations (spina bifida, encephalocele, anencephalus, omphalocele) were detected when both methods were used. Screening by routine ultrasound alone detected only four malformations and by measurement of alpha-fetoprotein alone only seven. The results suggest that, in a low risk population, ultrasound should be combined with the measurement of alpha-fetoprotein in screening for neural tube defects. Measurement of alpha-fetoprotein is indispensable in detection of the small neural tube defects, where the fetus would survive with severe sequelae. The semi-quantitative analysis of alpha-fetoprotein that may be used in combination with ultrasound examination is of negligible cost.

A zinc-dependent peptidase in prostatic organelles present in seminal plasma.

The electrophoretic seminal plasma protein pattern changes obviously within a few hours if the plasma is not frozen after the ejaculation. Mainly cathodal proteins disappear. Addition of serine protease inhibitors has no apparent affect, but o-phenanthroline markedly retards the conversions seen on electrophoresis. The subcellular organelles in seminal plasma are shown to contain a zinc ion dependent peptidase with optimal activity at pH 7.8. Estimations of enzyme activity may be performed using the elastase substrates n-tert butyloxycarbonyl-L-alanine-paranitrophenylester (Boc(Ala)) and succinyl(alanine)3-paranitroanilide (Suc(Ala)3pNA). The enzyme is inactivated by addition of o-phenanthroline and is completely reactivated by the addition of zinc ions.