Glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are glycoproteins secreted by prostate epithelial cells, and have a long clinical history of use as serum biomarkers of prostate cancers.

These two proteins are present at significantly higher concentrations in seminal plasma, making this proximal fluid of the prostate a good source for purifying enough protein for characterization of prostate disease associated changes in glycan structures. With the use of seminal fluid samples representative of normal control, benign prostatic disease and prostate cancers, PAP and PSA were enriched by thiophilic absorption chromatography.

Released N-linked glycan constituents from both proteins were analyzed by a combination of normal phase HPLC and MALDI-TOF spectrometry. For PSA, 40 putative glycoforms were determined, and 21 glycoforms were determined for PAP. PAP glycans were further analyzed with a hybrid triple quadrupole/linear ion trap mass spectrometer to assign specific glycoform classes to each of the three N-linked sites. The glycans identified in these studies will allow for more defined targeting of prostate disease-specific changes for PAP, PSA and other secreted prostatic glycoproteins.

White KY, Rodemich L, Nyalwidhe JO, Comunale MA, Clements MA, Lance RS, Schellhammer PF, Mehta AS, Semmes OJ, Drake RR.
Department of Microbiology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA

Prostatic acid phosphatase, a neglected ectonucleotidase.

Two recent papers reveal that the soluble and secreted prostatic acid phosphatase, an enzyme that has long served as a diagnostic marker for prostate cancer, has a membrane-bound splice variant. This enzyme exhibits ecto-5'-nucleotidase activity, is widely distributed, and implicated in the formation of chronic pain.

While prostatic acid phosphatase hydrolyzes phosphomonoesters other than 5'-nucleoside monophosphates these novel data suggest that, in addition to ecto-5'-nucleotidase and the alkaline phosphatases, prostatic acid phosphatase must be taken into account in future studies on extracellular adenosine production.

Institute of Cell Biology and Neuroscience, Biocenter, Goethe-University, 60438, Frankfurt, Germany,

Catalytic behaviour of acid phosphatase immobilized on natural supports in the presence of manganese or molybdenum

Synthetic complexes were formed by interaction between acid phosphatase and either tannic acid or natural allophanic clay, and used as model systems to simulate enzymatic reactions occurring in heterogeneous environment. The presence of Mn and Mo on the kinetic constants of the immobilized phosphatase was also tested. Complexes were prepared at 30 °C by interaction of acid phosphatase and tannic acid or allophanic clay in the presence and absence of Mn or Mo. The effect of the addition order of the metal (micronutrient in soils) to the clay–enzyme complexes was evaluated, as well. acid phosphatase immobilized on tannic acid and mineral clay showed variable activity levels. When phosphatase was immobilized on tannic acid, a recovery of about 51% of the initial enzymatic activity and a decrease of 17% of its catalytic efficiency was measured. The presence of Mn and Mo decreased the Vmax of phosphatase–tannic complexes as compared with that of the free phosphatase. All the added phosphatase molecules were immobilized onto the clay, when it was used as the immobilizing support. Phosphatase–clay complexes showed an increase of both enzymatic activity (higher Vmax value) and substrate affinity (lower Km values) as compared to the free enzyme, resulting in an increase by about 65% of the catalytic efficiency. The presence of Mn added at the same time with enzyme and clay decreased the enzymatic activity of the immobilized enzyme. However, when Mn was applied after the interaction of the enzyme with the clay, no significant effects on the residual activity of the immobilized enzyme were observed. Conversely, the order of the addition of Mo to the clay–enzyme complexes strongly influenced the activity and the kinetic behaviour of the immobilized enzyme. Moreover, Mo had higher inhibitory effects than Mn on phosphatase immobilized on both supports. The overall results seem to suggest that the immobilization of acid phosphatase on clay not only preserved but also enhanced the activity of the enzyme as compared with organic matter.

ARTICLE

Significance of Serum Tartrate-Resistant Acid Phosphatase in Rheumatoid Arthritis.

Abstract Human serum contains two related isoforms of tartrate-resistant acid phosphatase namely TRACP 5a and TRACP 5b. Serum TRACP 5a protein is increased in about 1/3 of RA sera. This study was undertaken to examine the significance of serum TRACP isoforms 5a and 5b as disease markers of inflammation and bone destruction in rheumatoid arthritis (RA). 118 patients were recruited including 50 with RA (25 with nodules), 26 with osteoarthritis (OA) and 42 with other rheumatic diseases. 26 healthy adults served as controls. Serum TRACP-5a activity, TRACP-5a protein and TRACP-5b activity were determined by in-house immunoassays. C-reactive protein (CRP) was determined by in-house immunoassay using commercial antibodies and CRP. Other commercial markers included bone-specific alkaline phosphatase (bALP), C-telopeptides of type-I collagen (ICTP), cartilage glycoprotein-39 (YKL-40), and IgM rheumatoid factors (IgM-RF). Mean TRACP-5a protein was significantly elevated only in RA compared to healthy control and other disease groups. TRACP 5a protein correlated significantly only with IgM-RF in RA. Among RA patients, mean TRACP 5a protein and IgM RF were significantly higher in nodule formers. In contrast, TRACP-5b activity was slightly elevated in RA and correlated with bALP. ICTP and YKL-40, but not with IgM-RF or CRP. Mean TRACP-5b activity was no different in RA patients with or without nodules. TRACP isoforms could be useful disease markers in RA: TRACP-5a protein may be a measure of systemic inflammatory macrophage burden and disease severity. TRACP-5b activity is a marker for osteoclast number and perhaps local or systemic bone destruction.

SH3BP2 is an Activator of NFAT Activity and Osteoclastogenesis

Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLCγ1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

Steven A. Lietmana, b, Lihong Yina, b and Michael A. Levinec

aDepartment of Orthopaedic Surgery, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

bDepartment of Biomedical Engineering, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA

cThe Division of Endocrinology, The Children’s Hospital of Philadelphia, 34th & Civic Center Boulevard, Philadelphia, PA 19104, USA

ARTICLE

Utility of serum tartrate-resistant acid phosphatase (TRACP5b) as a bone resorption marker in patients with chronic kidney disease: independence from

Background: Serum tartrate-resistant acid phosphatase (TRACP) 5b levels were assessed in predialysis patients with chronic kidney disease (CKD). The aim of the study was to establish the usefulness of a new assay for TRACP5b in assessing bone turnover in these patients.

Methods: Serum concentrations of two bone resorption markers, TRACP5b and N-Terminal cross-linking telopeptide of type I collagen (NTX-I); two bone formation markers, bone specific alkaline phosphatase (Bone ALP) and intact osteocalcin (OC[1-49]); and parathyroid hormone (PTH) were measured in 98 predialysis CKD patients.

Results: Log serum TRACP5b and other bone markers were significantly negatively correlated with glomerular filtration rate (GFR) and positively correlated with log serum PTH, suggesting an increase in serum bone markers with development of secondary hyperparathyroidism.

Multiple regression analysis including age, gender, BMI, the presence of diabetes, GFR, and log serum PTH showed an association of log serum PTH with log serum TRACP5b and other bone markers. GFR was associated with log serum NTX-I and log OC[1-49], but not with log serum TRACP5b or log Bone ALP. These data show that renal dysfunction does not influence serum TRACP5b and Bone ALP, but has an influence on NTX-I and OC[1-49].

Conclusion: Serum TRACP5b may be a good marker for serum bone resorption in predialysis CKD patients, since it is not affected by renal dysfunction.

Yamada S, Inaba M, Kurajoh M, Shidara K, Imanishi Y, Ishimura E, Nishizawa Y.
Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.Clin Endocrinol (Oxf). 2008 Jan 23

Acid Phosphatase -semen chemicals promotes HIV infections

HIV 's ability to infect human cells may be vastly enhanced by the presence of a chemical that occurs naturally in semen , research suggests.

The discovery could offer a new target for preventing the spread of AIDS , for example, by incorporating potential inhibitors of the semen chemical into the powerful new microbicides currently being developed to halt the spread of the virus
While searching for chemicals in semen that could block HIV transmission, an international team led by Frank Kirchhoff at the University Clinic of Ulm in Germany, was instead surprised to find that the naturally occurring chemical prostatic acidic phosphatase (PAP) promoted HIV infection.

Closer inspection revealed that PAP formed tiny fibres known as amyloid fibrils that were able to "ferry" the virus into the immune cells that it preys upon.

"Most enhancers have maybe a two or three-fold effect, but here the effect was amazing – more than 50-fold and, under certain conditions, more than 100,000-fold," says Kirchhoff. "At first, I didn't believe it, but we ran the experiment over and over, always with the same result."

Virus 'ferry'
"The fibrils act like a ferry. They pick the viruses up and then bring them to the cell," adds fellow researcher Wolf-Georg Forssmann at the Hannover Medical School, also in Germany.

Significantly, their laboratory tests showed that the fibrils' ability to assist infection was greatest when the levels of infectious virus were low – resembling the conditions in which sexual HIV-1 transmission usually occurs.

HIV-1, the most common and virulent form of the virus that causes AIDS, has infected about 60 million people and caused over 20 million deaths. Globally, most infections result from genital exposure to the semen of HIV-positive men.

Women who acquire HIV-1 through vaginal intercourse constitute almost 60% of new infections in Africa. Therefore the latest findings could have enormous clinical and public health implications, particularly for regions such as Sub-Saharan Africa where AIDS has caused devastation.

'Important discovery'
Kirchhoff says he hoped that agents that blocked prostatic acidic phosphatase might be developed and added to vaginal microbicides that women could use to protect themselves from infection. He also believes the discovery could have important implications for researchers hunting an elusive HIV vaccine.

Jonathan Weber, an eminent HIV researcher at Imperial College London , UK, was positive about the findings. "Kirchhoff is a great scientist and this is meticulous work," he says.

But Weber says there is much work to be done before new microbicides could be produced as a result of the findings. He notes, too, that the ability of HIV-positive women to infect men suggested that the presence of seminal PAP fibrils was not always required for infection to occur.

He adds, however: "This work is exciting because it opens up whole new avenues of prevention. It really does suggest entirely new directions of research, and like many of the really important discoveries it was completely unexpected."

Journal reference: Cell (DOI: 10.1016/j.cell.2007.10.014)

HIV and AIDS – Learn more about the worst pandemic in human history in our continuously updated special report.

Preliminary investigations on the standardization and quality control for the determination of acid phosphatase activity in seminal plasma

Background
The determination of Acid Phosphatase ACP in seminal plasma was considered as an appropriate biochemical marker to evaluate prostate function , as recommended by the WHO manual. However, few reports on the standardization and quality control for the determination of biochemical markers in seminal plasma have been documented.

Methods
Two frozen samples of seminal plasma with or without phenylmethylsulfonyl fluoride were determined for their Acid Phosphatase (ACP) levels. The Acid Phosphatase level and sperm concentration of each of 72 samples of seminal plasma obtained at 1000×g for 10 min or 3000×g for 15 min centrifugation were assayed. Acid Phosphatase activity in 10 samples of seminal plasma was measured immediately or standing for 30 min after dilution. The ACP levels in seminal plasma with or without chymotrypsin were also assayed.

Results
There was no significant difference of ACP levels (P = 0.166) but of sperm concentrations (P = 0.000) in seminal plasma obtained by centrifugation at different velocity. ACP activities in seminal plasma measured when standing for 30 min after dilution were significantly lower than those measured immediately after dilution (P = 0.001). Both chymotrypsin and freezing–thawing had no apparent effect on the determination of ACP in seminal plasma.

Conclusion
The results indicated that standing time after dilution and centrifugation velocity should be standardized, and frozen seminal plasma could serve as the quality control products for the determination of ACP activity among different laboratories.

Department of Reproduction and Genetics, Jinling Hospital, School of Medicine, Nanjing University , Nanjing, Jiangsu 210002, China
Clinica Chimica Acta
Volume 375, Issues 1-2, January 2007, Pages 76-81

Acid Phospatase

Acid phosphatase is a phosphatase, a type of enzyme, used to free attached phosphate groups from other molecules during digestion. Acid Phosphatase is basically a phospho homo esterase . Acid Phosphatase is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acid pH optimum.

Different forms of acid phosphatase are found in different organs, and their serum levels are used as a diagnostic for disease in the corresponding organs. For example, elevated prostatic acid phosphatase levels may indicate the presence of prostate cancer.

Prostatic acid phosphatase (PAP) is an enzyme produced by the prostate. Acid phosphatase may be found in increased amounts in men who have prostate cancer or other diseases.

The highest levels of acid phosphatase are found in metastasized prostate cancer. Diseases of the bone, such as Paget's disease or hyperparathyroidism, diseases of blood cells, such as sickle-cell disease or multiple myeloma or lysosomal storage diseases, such as Gaucher's disease, will show moderately increased levels.