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Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Chemistry.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Sanford Burnham Medical Research Institute, CA USA .
Sanford Burnham Medical Research Institute, CA USA .
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2013 (English)In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, Vol. 53, no 2, 399-408 p.Article in journal (Refereed) Published
Abstract [en]

Mouse serum alkaline phosphatase (ALP) is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP isozymes and isoforms. Mouse ALP was extracted from liver, kidney, intestine, and bone from vertebra, femur and calvaria tissues. Serum from mixed strains of wild-type (WT) mice and from individual ALP knockout strains were investigated, i.e., Alpl(-/-) (a.k.a. Akp2 encoding tissue-nonspecific ALP or TNALP), Akp3(-/-) (encoding duodenum-specific intestinal ALP or dIALP), and Alpi(-/-) (a.k.a. Akp6 encoding global intestinal ALP or gIALP). The ALP isozymes and isoforms were identified by various techniques and quantified by high-performance liquid chromatography. Results from the WT and knockout mouse models revealed identical bone-specific ALP isoforms (B/I. B1, and B2) as found in human serum, but in addition mouse serum contains the B1x isoform only detected earlier in patients with chronic kidney disease and in human bone tissue. The two murine intestinal isozymes, dIALP and gIALP, were also identified in mouse serum. All four bone-specific ALP isoforms (B/I, B1x, B1, and B2) were identified in mouse bones, in good correspondence with those found in human bones. All mouse tissues, except liver and colon, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Histochemical staining, Northern and Western blot analyses confirmed undetectable ALP expression in liver tissue. ALP activity staining showed some positive staining in the bile canaliculi for BALB/c and FVB/N WT mice, but not in C57BI/6 and ICR mice. Taken together, while the main source of ALP in human serum originates from bone and liver, and a small fraction from intestine (andlt;5%), mouse serum consists mostly of bone ALP, including all four isoforms, B/I, B1x, B1, and B2, and two intestinal ALP isozymes dIALP and gIALR We suggest that the genetic nomenclature for the Alpl gene in mice (i.e., ALP liver) should be reconsidered since murine liver has undetectable amounts of ALP activity. These findings should pave the way for the development of user-friendly assays measuring circulating bone-specific ALP in mouse models used in bone and mineral research.

Place, publisher, year, edition, pages
Elsevier , 2013. Vol. 53, no 2, 399-408 p.
Keyword [en]
Alkaline phosphatase, Bone, Glycosylation, Hypophosphatasia, Knockout mice, Mineralization
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-90744DOI: 10.1016/j.bone.2012.12.048ISI: 000315763700010OAI: oai:DiVA.org:liu-90744DiVA: diva2:614709
Note

Funding Agencies|County Council of Ostergotland in Sweden||National Institutes of Health, USA|DE012889|

Available from: 2013-04-05 Created: 2013-04-05 Last updated: 2017-12-06
In thesis
1. Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis
Open this publication in new window or tab >>Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human skeleton is a living and dynamic tissue that constantly is being renewed in a process called bone remodeling. Old bone is resorbed by osteoclasts and new bone is formed by osteoblasts. Bone is a composite material made up by mineral crystals in the form of hydroxyapatite (calcium and phosphate) that provides the hardness of bone, and collagen fibrils that provides elasticity and flexibility. Alkaline phosphatase (ALP) is a family of enzymes that is present in most species and catalyzes the hydrolysis of various phosphomonoesters at alkaline pH. Despite the generalized use of ALP as a biochemical marker of bone formation, the precise function of bone ALP (BALP) is only now becoming clear. Three circulating human BALP isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue.

Paper I. Three endogenous phosphocompounds, (i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP) and phosphoethanolamine (PEA)), have been suggested to serve as  physiological substrates for BALP. The BALP isoforms display different catalytic properties towards PPi and PLP, which is attributed to their distinct N-linked glycosylation patterns. The catalytic activity, using PEA as substrate, was barely detectable for all BALP isoforms indicating that PEA is not a physiological substrate for BALP.

Paper II. Mouse serum ALP is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP. We characterized the circulating and tissue-derived mouse ALP isozymes and isoforms from mixed strains of wild-type and knockout mice. All four BALP isoforms (B/I, B1x, B1, and B2) were identified in mouse serum and bone tissues, in good correspondence with those found in human bones. All mouse tissues, except liver, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP.

Paper III. The objective of this study was to investigate the binding properties of human collagen type I to human BALP, including the two BALP isoforms B1 and B2, together with ALP from human liver, human placenta and E. coli. A surface plasmon resonance-based analysis showed that BALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. The B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform, indicating that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I.

Paper IV. Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and frequently used as a marker of bone resorption. Intriguingly, recent studies show that TRAP is also expressed in osteoblasts and osteocytes. TRAP displays enzymatic activity towards the endogenous substrates for BALP, i.e., PPi and PLP. Both TRAP and BALP can alleviate the inhibitory effect of osteopontin on mineralization by dephosphorylation, which suggests a novel role for TRAP in skeletal mineralization.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. 66 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1511
National Category
Clinical Medicine Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomaterials Science Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-127100 (URN)10.3384/diss.diva-127100 (DOI)978-91-7685-824-0 (ISBN)
Public defence
2016-05-13, Berzeliussalen, Campus Valla, Linköping, 09:00 (Swedish)
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Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-05-02Bibliographically approved

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Linder, CeciliaEnglund, UlrikaMagnusson, Per

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