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Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
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: urn:nbn:se:liu:diva-127100DOI: 10.3384/diss.diva-127100ISBN: 978-91-7685-824-0 (print)OAI: oai:DiVA.org:liu-127100DiVA: diva2:919470
Public defence
2016-05-13, Berzeliussalen, Campus Valla, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-05-02Bibliographically approved
List of papers
1. Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.
Open this publication in new window or tab >>Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.
2009 (English)In: Bone, ISSN 1873-2763, Vol. 45, no 5, 987-993 p.Article in journal (Refereed) Published
Abstract [en]

Three circulating human bone alkaline phosphatase (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. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD(10) isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21351 (URN)10.1016/j.bone.2009.07.009 (DOI)19631305 (PubMedID)
Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2016-04-14
2. Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
Open this publication in new window or tab >>Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
Show others...
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
Keyword
Alkaline phosphatase, Bone, Glycosylation, Hypophosphatasia, Knockout mice, Mineralization
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-90744 (URN)10.1016/j.bone.2012.12.048 (DOI)000315763700010 ()
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
3. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I
Open this publication in new window or tab >>Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I
2016 (English)In: Calcified Tissue International, ISSN 0171-967X, E-ISSN 1432-0827, Vol. 98, no 3, 284-293 p.Article in journal (Refereed) Published
Abstract [en]

Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2016
Keyword
Alkaline phosphatase; Bone; Collagen; Glycosylation; Mineralization; Surface plasmon resonance
National Category
Endocrinology and Diabetes Dentistry
Identifiers
urn:nbn:se:liu:diva-127099 (URN)10.1007/s00223-015-0088-0 (DOI)000373744700008 ()26645431 (PubMedID)
Note

Funding agencies:  Region Ostergotland, Sweden

Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-11-30Bibliographically approved

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