Glycosylation-deficient mutations in tissue-nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia


Tissue-nonspecific alkaline phosphatase (TNSALP) is a membrane glycoprotein with a proposed role in bone mineralization. Indeed, mutations in TNSALP have been identified in patients with hypophosphatasia (HPP), a genetic disease characterized by hypomineralization of bone and teeth and a deficiency in serum ALP activity. TNSALP has five potential N-glycosylation sites at N140, N230, N271, N303 and N430 by standard nomenclature.

A mutation at one of these sites, N430, was recently detected in a patient with infantile HPP. Using site-directed mutagenesis, we demonstrated that TNSALP has five N-glycans in transfected COS-1 cells and that individual single N-glycan deletion mutants of TNSALP retain the dimeric structure required for ALP activity, excluding the possibility that any single N-glycan plays a vital role in the structure and function of TNSALP.

However, we found that TNSALP (N430Q) and TNSALP (N430E) mutants, but not a TNSALP (N430D) mutant, failed to form dimers. The TNSALP (N430S) mutant linked to infantile HPP was glycosylation-defective and unable to dimerise, similar to TNSALP (N430Q) and TNSALP (N430E) mutants; therefore, TNSALP (N430S) was established as a severe allele without strong ALP activity. By contrast to individual single N-glycan deletion mutants, TNSALP devoid of all five N-glycans was present to a much lesser extent than wild-type TNSALP in transfected cells, possibly reflecting its instability.

A comprehensive analysis of a series of multiple N-glycan depletion mutants in TNSALP revealed that three N-glycans on N230, N271 and N303 were the minimal requirement for the structure and function of TNSALP and a prerequisite for its stable expression in a cell.



Enzymatically mediated bioprecipitation of heavy metals from industrial wastes and single ion solutions by mammalian alkaline phosphatase


J Environ Sci Health A Tox Hazard Subst Environ Eng. 2013;48(1):79-85. doi: 10.1080/10934529.2012.707851

The study was aimed at investigating the potential use of calf intestinal alkaline phosphatase (CIAP) enzyme in the removal of heavy metals (Cd(2+), Ni(2+), Co(2+) and Cr(3+/6+)) from single ion solutions as well as tannery and electroplating effluents. CIAP mediated bioremediation (white biotechnology) is a novel technique that is eco-friendly and cost effective unlike the conventional chemical technologies. Typical reactions containing the enzyme (CIAP) and p-nitrophenyl phosphate (pNPP) as substrate in Tris-HCl buffer (pH 8 and 11) and either single ion metal solutions (250 ppm and 1000 ppm) or effluents from tannery or electroplating industry were incubated at 37°C for 30 min, 60 min and 120 min. The inorganic phosphate (P(i)) generated due to catalytic breakdown of pNPP complexes free metal ions as metal-phosphate and the amount of metal precipitated was derived by estimating the reduction in the free metal ion present in the supernatant of reactions employing atomic absorption spectrophotometer (AAS). Better precipitation of metal was obtained at pH 11 than at pH 8 and between the two concentrations of different metals tested, an initial metal concentration of 250 ppm in the reaction gave more precipitation than with 1000 ppm. Experimental data showed that at pH 11, the percentage of removal of metal ions (for an initial concentration of 250 ppm) was in the following order: Cd(2+) (80.99%) > Ni(2+) (64.78%) > Cr(3+) > (46.15%) > Co(2+) (36.47%) > Cr(6+) (32.33%). The overall removal of Cr(3+) and Cr(6+) from tannery effluent was 32.77% and 37.39% respectively in 120 min at pH 11. Likewise, the overall removal of Cd(2+), Co(2+) and Ni(2+) from electroplating effluent was 50.42%, 13.93% and 38.64% respectively in 120 min at pH 11. The study demonstrates that bioprecipitation by CIAP may be a viable and environmental friendly method for clean-up of heavy metals from tannery and electroplating effluents

Chaudhuri G, Shah GA, Dey P, S G, Venu-Babu P, Thilagaraj WR.

Sourcea Department of Biotechnology, School of Bioengineering , SRM University , Kattankulathur , Tamil Nadu , India

Alkaline phosphatase normalization is associated with better prognosis in primary sclerosing cholangitis

Primary sclerosing cholangitis results in elevated but fluctuating serum alkaline phosphatase levels that occasionally return to normal.

To investigate the frequency of normalization of alkaline phosphatase in newly diagnosed primary sclerosing cholangitis patients and the subsequent clinical outcomes.

Records of newly diagnosed primary sclerosing cholangitis patients were examined retrospectively for laboratory values and clinical end points (cholangiocarcinoma, liver transplantation and death) within 10 years of diagnosis. Data from a recent prospective ursodeoxycholic acid treatment trial were also studied.

Eighty-seven patients met the inclusion criteria. Normalization of alkaline phosphatase was seen in 35 (40%) patients. Five (14%) patients with normalization reached an end point whereas 17 (33%) of the patients with persistent elevation reached an end point (P = 0.02). Ursodeoxycholic acid was used similarly by both groups. When the investigative criteria were applied to a prospective trial, there was again a significant relationship between normalization of alkaline phosphatase and survival in patients receiving ursodeoxycholic acid (P < 0.01) and the placebo group (P = 0.02).

Serum alkaline phosphatase was found to normalize in a high proportion of newly diagnosed primary sclerosing cholangitis patients. This was significantly associated with a better prognosis in a retrospective cohort and when data from a prospective treatment trial was evaluated

Digestive and Liver Disease
Volume 43, Issue 4, April 2011, Pages 309-313

Serum alkaline phosphatase changes predict survival independent of PSA changes in men

U.S. Oncology Research, Inc., Texas Oncology and Baylor College of Medicine, Webster, TX 77598, USA.

The association of a change in serum alkaline phosphatase (ALP) with overall survival OS in men with metastatic castration-resistant prostate cancer (CRPC) receiving chemotherapy is unknown. We evaluated the association of changes in ALP within 90 days with OS in men with CRPC and bone metastases treated with docetaxel or mitoxantrone in the TAX327 trial.

Eligible patients included those with bony metastatic disease, baseline ALP ≥ 120 u/L (upper limit of normal) and ≥2 post-therapy measurements of ALP available. Survival was estimated using the Kaplan-Meier method and prognostic potential of change in ALP was evaluated using Cox proportional hazards regression. Surrogacy was calculated by the Likelihood Reduction Factor.

601 patients met the eligibility criteria. By day 90, 159 patients had ALP normalization (< 120 u/L) and 442 patients did not normalize. Normalization of ALP remained prognostic for OS after adjusting for PSA decline ≥ 30% by day 90 (HR 0.79, 95% CI = 0.65-0.97, P = 0.022). Increase in ALP remained prognostic for OS when adjusting for PSA increase ≥ 50% by day 90 (HR 1.69, 95% CI = 1.33-2.14, P < 0.001). ALP changes did not meet criteria for surrogacy for OS.

For men with CRPC, bone metastasis and high baseline ALP receiving docetaxel or mitoxantrone chemotherapy, normalization of ALP by day 90 was predictive of better survival independent of ≥30% PSA declines. An increase in ALP by day 90 was also predictive of poor survival independent of ≥50% PSA increase. Given the ready availability of ALP, the validation of our data is warranted.

Written by:
Sonpavde G, Pond GR, Berry WR, de Wit R, Armstrong AJ, Eisenberger MA, Tannock IF


Alkaline Phosphatase May Be a Marker of Inflammation in CKD Patients

ORLANDO, Fla.—Serum alkaline phosphatase may be a marker for inflammation in CKD patients, according to data presented here at the National Kidney Foundation's 2010 Spring Clinical Meetings.

In a study of more than 900 adults with CKD (mean age 69.6 years, 37% men, 7% African-American) who participated in the Third National Health and Nutrition Examination Survey, researchers at the University of Utah School of Medicine in Salt Lake City found that higher serum alkaline phosphatase levels were associated with lower levels of serum 25-hydroxyvitamin D (25-D). Subjects in the highest quartile of serum alkaline phosphatase had a 2.54-fold higher odds of elevated C-reactive protein levels after adjusting for serum 25-D levels. “Therefore, serum alkaline phosphatase might be a marker of inflammatory milieu in the CKD population,” the investigators concluded.

By: Jody A. Charnow


Tumor necrosis factor-α increases alkaline phosphatase expression in vascular smooth muscle cells via MSX2 induction

Hye-Lim Leea, Kyung Mi Wooa, Hyun-Mo Ryooa and Jeong-Hwa Baek, a,

a Department of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea

Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-α (TNF-α) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-α-mediated vascular calcification has not yet been fully defined. Therefore, in this study, we aimed to investigate whether MSX2 acts as a crucial regulator in TNF-α-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs).

TNF-α increased the expression of osteogenic marker genes including RUNX2, osterix, alkaline phosphatase (ALP), and bone sialoprotein, and it also promoted matrix mineralization in VSMCs. In addition, TNF-α enhanced MSX2 expression in a dose- and time-dependent manner. MSX2 over-expression alone induced ALP expression, whereas knockdown of MSX2 with small interfering RNA completely blocked TNF-α-induced ALP expression.

New protein synthesis was dispensable for MSX2 induction by TNF-α, and the inhibition of NF-κB by BAY-11-7082 or by dominant negative IκBα abolished the TNF-α-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression.

In conclusion, our study suggests that TNF-α directly induces MSX2 expression through the NF-κB pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs


Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients

Objective: Alkaline phosphatase (AP) attenuates inflammatory responses by lipopolysaccharide detoxification and may prevent organ damage during sepsis. To investigate the effect of AP in patients with severe sepsis or septic shock on acute kidney injury.

Design and Setting: A multicenter double-blind, randomized, placebo-controlled phase IIa study (2:1 ratio).

Patients: Thirty-six intensive care unit patients (20 men/16 women, mean age 58 ± 3 years) with a proven or suspected Gram-negative bacterial infection, ≥2 systemic inflammatory response syndrome criteria (<24 hours), and <12 hours end-organ dysfunction onset were included.

Intervention: An initial bolus intravenous injection (67.5 U/kg body weight) over 10 minutes of AP or placebo, followed by continuous infusion (132.5 U/kg) over the following 23 hours and 50 minutes.

Measurements and Main Results: Median plasma creatinine levels declined significantly from 91 (73-138) to 70 (60-92) μmol/L only after AP treatment. Pathophysiology of nitric oxide (NO) production and subsequent renal damage were assessed in a subgroup of 15 patients. A 42-fold induction (vs. healthy subjects) in renal inducible NO synthase expression was reduced by 80% ± 5% after AP treatment. In AP-treated patients, the increase in cumulative urinary NO metabolite excretion was attenuated, whereas the opposite occurred after placebo. Reduced excretion of NO metabolites correlated with the proximal tubule injury marker glutathione S-transferase A1-1 in urine, which decreased by 70 (50-80)% in AP-treated patients compared with an increase by 200 (45-525)% in placebo-treated patients.

Conclusions: In severe sepsis and septic shock, infusion of AP inhibits the upregulation of renal inducible NO synthase, leading to subsequent reduced NO metabolite production, and attenuated tubular enzymuria. This mechanism may account for the observed improvement in renal function.

Critical Care Medicine:
February 2009 - Volume 37 - Issue 2 - pp 417-e1Heemskerk, Suzanne PhD; Masereeuw, Rosalinde PhD; Moesker, Olof; Bouw, Martijn P. W. J. M.; van der Hoeven, Johannes G. MD, PhD; Peters, Wilbert H. M. PhD; Russel, Frans G. M. PhD; Pickkers, Peter MD, PhD; on behalf of the APSEP Study Group