Jukka Hellman

Biochemical Markers of Bone Metabolism and Prediction of Fracture in Elderly Women

We studied the ability of various markers of bone turnover to predict fracture in 1040 randomly recruited 75‐year‐old women. A total of 178 of the women sustained at least one fracture during follow‐up (mean, 4.6 years). In elderly women, TRACP5b and urinary fragments of osteocalcin are promising new markers for prediction of fracture, in particular, vertebral fracture.

Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the 32P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

Enzymatic action of prostate-specific antigen (PSA or hk3) : Substrate specificity and regulation by Zn2+, a tight-binding inhibitor

In semen, prostate‐specific antigen (PSA or hK3) digests the gel proteins semenogelin I and II, resulting in liquefaction and the release of motile spermatozoa. We characterized the substrate specificity and zinc‐mediated inhibition of PSA.

Two‐Site Immunoassays for Osteoclastic Tartrate‐Resistant Acid Phosphatase Based on Characterization of Six Monoclonal Antibodies

Tartrate‐resistant acid phosphatase (TRAP), an enzyme expressed in bone‐resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two‐site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4°C and −20°C or five thawing‐freezing cycles, did not change the TRAP concentration detected using the two‐site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1–5C1 and O1A–J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1–5C1 and O1A–J1B may be useful in determining the bone resorption rate.

Identification of novel proteolytic forms of osteocalcin in human urine

In this study, we report the isolation and characterization of osteocalcin in human urine using mass spectrometry and N-terminal sequencing. Multiple proteolytic forms of osteocalcin were found, which consisted of 16-27 residues from the middle region of the molecule. Several fragments had residue Gly7 at the N-terminus and the most predominant was fragment 7-31. Additional fragments starting from residue Asp14 were detected in the samples of children and young adults. Immunochemical detection of urine osteocalcin fragments had a statistically significant negative correlation to bone mineral density in evaluation of urine samples from 75-year-old women. Thus, the measurement of osteocalcin fragments in urine may have potential applications in diagnostics related to disorders of bone metabolism.

A mouse molecular mimic of human vascular adhesion protein-1

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.

Development of an immunoassay for the detection of cystatin C dimers

Human cystatin C (CysC) is a reversible cysteine protease inhibitor, which is abundantly secreted to body fluids. It is a potential marker of kidney dysfunction, but has been suggested to be of diagnostic importance in a number of neurodegenerative diseases, as well. The amyloid formation by a L68Q variant CysC accounts for the hereditary CysC amyloid angiopathy (HCCAA). Also, the wild type CysC forms inactive dimers at partly denaturing conditions through a domain swapping mechanism. Here, we have developed an immunoassay for the detection of dimeric CysC consisting of either a full length or an N-terminally truncated form. A codon optimized gene encoding a full length CysC was expressed in Escherichia coli, where the product was directed to the periplasmic space. Two different forms of CysC were isolated, a full length product and a form proteolytically truncated by 8 N-terminal amino acid residues. In vitro dimerization experiments were conducted in order to enable the selection of monoclonal antibodies for the construction of an immunoassay being able to primarily recognize the dimers. The analytical detection limit of the assay was 0.043 microg/l, with assay imprecision below 16%. The assay was linear in the range of 5-100 microg/l (R(2)=0.997). The dimer assay was employed for the measurement of serum and cerebrospinal fluid (CSF) sample panel of 20 multiple sclerosis (MS) and 22 non-MS patients. A dimer signal was observed in both serum and CSF samples. The dimer signals from CSF were approximately 2-22 times higher (average 13) than the corresponding signals from serum samples. However, the measured signal levels between the different patient groups showed no statistically significant difference in serum or in CSF (P=0.07 and P=0.98 respectively). In conclusion, the immunoassay provides direct means for detecting CysC dimers in serum and CSF in respect to the amount of total CysC.

Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase

A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.

Stabilization of thin-layer agarose gels after isoelectric focusing with polyacrylamide enables reverse imidazole-zinc staining and facilitates two-dimensional gel electrophoresis.

Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated.